Ординатура / Офтальмология / Английские материалы / Mechanisms of the Glaucomas_Shields, Tombran-Tink, Barnstable_2008

IV

MOLECULAR AND CELLULAR RESPONSES

IN THE EYE TO GLAUCOMA

INTRODUCTION

The earlier sections have described the incidence of glaucomas, the role of elevated intraocular pressure, and some of the insights that genetic studies are giving into the causes and classification of this disease. It is clear that multiple causes may give rise to the common effect of ganglion cell death. The purpose of this part is to examine some of the cellular and molecular changes that occur in the eye in glaucoma.

As changes in aqueous outflow are responsible for many forms of glaucoma, it is appropriate for this part to start with a discussion of the structures involved in aqueous production and flow and the pathological changes that can alter this physiological process.

The role of glial cells is often overlooked in neurodegenerative diseases. In this part, changes in specific glial populations in the optic nerve head as well as more general changes in astrocytes and microglia are discussed. It is clear that physical changes in the glia themselves, as well as the molecules they secrete, are critical for understanding the pathogenesis of glaucoma.

New methods of biochemical analysis allow the detection of changes among a large array of molecules in any tissue in a disease or following any experimental manipulation. These techniques have been applied to glaucoma and we now have large sets of data that describe the changes in specific RNAs or proteins that occur in various eye regions in the disease. These sets of data hold the promise of telling us how specific gene defects or ocular insults are translated into pathological processes that result in the neuropathies that we define as forms of glaucoma.

In this chapter, we consider the changes in aqueous humor dynamics and the outflow pathways of the eye that occur with age and glaucoma. The form of glaucoma referred to here is primary open-angle glaucoma (POAG), one age-related form of the disease in which intraocular pressure (IOP) is elevated and associated with an optic neuropathy. The trabecular meshwork (TM)-Schlemm’s canal region is a key modulator of aqueous outflow from the eye and hence of IOP (5). A second aqueous exit route is the uveoscleral pathway that plots a path through the ciliary muscle (CM) interstitium, suprachoroid, and sclera.

The TM is populated by cells which secrete and live in a relatively abundant extracellular matrix (ECM), forming a pathway through which aqueous passes to exit the eye. The exact modus operandi of these cells and their matrix is poorly understood. It is understood, however, that fewer cells populate this region with age and glaucoma and that their matrix environment changes (6–10). The chapter starts by outlining agerelated changes in aqueous physiology and the microstructure of the outflow pathways as observed in humans and monkeys. Comment is made on related changes found in glaucoma. We then discuss putative basic mechanisms of ageing and how they apply to the ageing TM and glaucoma.

CHANGES IN AQUEOUS HUMOR DYNAMICS

Intraocular Pressure

IOP remains relatively stable or increases slightly with age in many Western populations (11,12). Epidemiologic studies in the USA have shown an increase in IOP with age (13,14), whereas IOP in the Japanese decreases with age (15,16).

IOP in normal, healthy, free-ranging rhesus monkeys remains relatively constant with age, after an initial juvenile hypertensive phase (17–19). Cage-housed rhesus monkeys aged 3–29 years (human equivalent of 8–73 years), however, show a significant increase in IOP with age (20).

Outflow Facility

Total outflow facility, trabecular facility, and pseudofacility decrease with age in humans when measured by tonography or the perfusion of postmortem enucleated eyes (21–23). The age-related decrease in facility from the youngest (<40 years) to the oldest (>60 years) is about 30% (21,22). Measurement of outflow facility by the alternative method of fluorophotometry shows no difference with age. It is argued that fluorophotometric measurements are independent of pseudofacility and ocular rigidity (which increases with age) (24,25). In rhesus monkeys, however, baseline outflow facility measured by two-level constant pressure perfusion decreases with age (26,27). Atropine does not completely eliminate the age-related facility decline in monkeys, indicating the presence of atropine-independent, facility-relevant, and age-dependent changes in the TM, such as loss of cells or build-up of extracellular material (28).

In POAG, there is an age-related facility decrease similar in magnitude to that reported for non-glaucomatous patients (21). The absolute value of outflow facility in POAG is significantly less than in age-matched controls, however (29). A 10year longitudinal study of untreated ocular hypertension showed a progressive facility

decrease (30). Fluorophotometric outflow facility is significantly lower in ocular hypertension than in age-matched controls (31).

Uveoscleral Outflow

Early studies of uveoscleral outflow conducted in elderly humans with posterior segment tumors indicated that uveoscleral outflow accounts for no more than 25% of total aqueous outflow. In young healthy humans, uveoscleral outflow was calculated to account for 36% of total outflow but accuracy was in question as tonography was performed at low IOPs (32). More recently, uveoscleral outflow calculated from fluorophotometric measures of outflow facility and aqueous humor formation was determined to account for 54% of total aqueous drainage in young healthy humans, compared with 46% in older healthy subjects (25). Measured by isotope accumulation techniques, uveoscleral outflow in young healthy monkey eyes accounted for 45–70% of total aqueous drainage (33). In rhesus monkeys, uveoscleral outflow is about 50% lower in 25–29 year olds (equivalent to humans older than 60 years of age) compared with 3–10 year olds (20).

Uveoscleral outflow in POAG patients, as calculated from fluorophotometric measurements of aqueous humor flow and outflow facility, comprises 78% of total aqueous drainage, higher than in normal controls (34). Another study suggests, however, that uveoscleral outflow makes up 25% of aqueous humor drainage in eyes with ocular hypertension compared with 42% in normotensive controls (31). The different findings of these studies are difficult to reconcile and could be because of technical issues or measurement artifact. The exact role of uveoscleral outflow in glaucoma requires further study.

Aqueous Humor Formation

Tonographic and fluorophotometric studies in humans consistently show an agerelated decline in the rate of aqueous production of approximately 15–35% between the ages of 20 and 80 years (21–23,35). The decline probably occurs throughout adult life and is estimated to be 0.003–0.015 μl/min/year or 2.4%/decade (21,31,36,37). Some studies show a greater decline after 60 years of age, estimated at 1–2%/year or 0.025 μl/min/year (21,36). The age-related reduction in aqueous humor formation and increase in resistance to outflow counteract each other so that IOP elevation with age is not substantial.

The ultrafiltration component of aqueous humor formation is pressure sensitive and decreases with increasing IOP. This pressure-sensitive decrease in inflow, termed pseudofacility, mimics an increase in outflow facility as measured with tonography and constant pressure perfusion. Pseudofacility shows an age-dependent decline of 33% from 20–40 year-old to older 50–80+ year-old subjects (22,23).

The daytime rate of aqueous humor formation is similar in POAG and age-matched controls based on fluorophotometry in 20 POAG patients off therapy for 6 weeks (29). The night-time rate of aqueous humor formation is significantly higher in glaucoma than control subjects. In ocular hypertension, the rate of aqueous humor formation does not significantly change with age nor differ from age-matched controls (31).

ULTRASTRUCTURAL CHANGES WITH AGE AND GLAUCOMA

Trabecular Meshwork Cells

The number of human TM cells is estimated to decline with age at a rate of 0.58–0.85% per year (9,10,38). This is associated with more TM cell pigmentation, cell detachment from trabeculae, and increased fusion between adjacent trabeculae (10). The TM cell population declines from 1,200,000 cells at 20 years of age to approximately 500,000 cells by 80 years, reflecting an average loss of 12,000 cells per year. TM cell numbers in cynomolgus and rhesus monkeys also decrease with age (20,39).

In younger eyes, alpha-smooth muscle (sm) actin filaments are seen in nearly all cells of the TM. With increasing age, however, fewer cells stain for alpha-sm actin, and these are localized mainly in the posterior part of the TM and in the scleral spur. Human TM protein profiling also shows a decrease in G-actin with age, associated with increased type IV collagen. This suggests that a less contractile cellular phenotype develops with ageing, which could influence functioning of the outflow pathway. While cells expressing alpha-sm-actin decrease with age and glaucoma (40), alpha- sm-actin expression is induced by TGF-beta (41), which itself is increased in the aqueous of glaucomatous eyes (42). Perhaps by this means, an under-contractile and dysfunctional TM (eg., due to age) can be compensated for. Because TGF-beta is a complex multifunctional signaling molecule, however, its overexpression could alter the balance of signal transduction in the TM (e.g., in ECM) in ways that might ultimately raise IOP (43,44). Perfusing organ-cultured eyes with TGF-beta at supraphysiological levels decreases outflow facility (45). The relevance of TGF-beta to TM contractility, ageing, and glaucoma is still being worked out.

Trabecular Meshwork Extracellular Matrix

The cribriform plexus (network of elastic-like fibers in the cribriform area) connects with the endothelium of Schlemm’s canal by fine radially oriented fibrils. Anterior CM tendons are anchored within this plexus. CM contraction may thus influence the width of the cribriform layer and its intercellular spaces and so affect outflow resistance. The elastic-like fibers of the cribriform plexus are surrounded by a sheath of fine fibrils embedded in a nearly homogeneous matrix rich in chondroitin sulfates

(46–49).

In older human eyes (50–80 years), the elastic-like fibers in the cribriform meshwork become thicker and coarser, and there is increased plaque material associated with their sheaths (50,51). This could well affect outflow resistance and make it more difficult for the TM to sustain adequate outflow as its tone changes with age.

Histological analysis of the cribriform TM and inner wall of Schlemm’s canal of normal human eyes shows that its amorphous ECM comprises many major matrix proteins including collagen type IV, laminin, and fibronectin. mRNA for all three proteins is present in trabecular cells throughout the meshwork (52–55). Elastin localizes to the central core of sheath-derived plaques (55,56). Microfibrillarassociated glycoprotein (MAGP)-1, fibronectin, and fibrillin-1 localize mostly within

the peripheral mantle of the sheath surrounding the elastin core in sheath-derived plaques of the cribriform TM. The fibrillin-containing microfibrillar system may provide tensile strength and flexibility to the tissue (55,57). Because of its relationship with sheath-derived plaques and the cribriform region, it is suggested that the microfibrillar system, if dysfunctional, also plays a role in glaucoma pathogenesis. Myocilin is associated with the corneoscleral meshwork, cribriform TM, and microfibrillar architecture of sheath-derived plaques, but its significance to glaucoma is unclear (55,58). In what way ECM proteins change with age in the TM remains to be defined.

Ciliary Body

Ageing changes in the CM are significant as they can affect uveoscleral outflow. CM contraction also increases aqueous humor outflow by altering the configuration of the TM and Schlemm’s canal (59,60). In primate eyes, posterior CM tendons insert into Bruch’s membrane and the elastic network of the choroid (61). With age, collagen fibers of the choroid and sclera thicken, while elastic fibers of the choroid, Bruch’s membrane, and peripapillary region lose their elastin core and appear more electrondense (62,63). In the suprachoroidal layer and sclera, elastic-like fibers become thicker, have thicker sheaths, and more cross-linked microfibers merging at dense plates.

In older humans, the CM is short with a prominent inner edge of circular muscle fibers (64,65). It is theorized that the posterior elastic-like tendons lose their elasticity with age, preventing the muscle from being pulled backward during disaccommodation so that it retains its contracted appearance. Extracellular material between muscle bundles significantly increases with age, especially in the muscle’s intermediate reticular portion facing the anterior chamber. CM spaces continuous with the anterior chamber are also smaller.

CM plaques increase with age in correlation with the amount of Schlemm’s canal inner-wall plaques. At the CM tips, the elastic-like tendons and elastic-like fibers within the trabeculum ciliare undergo age-related changes similar to those described in the TM. The sheaths of the elastic-like fibers thicken markedly. Broad plates of banded collagen are seen where the tendon sheaths connect to the sheaths of the elastic-like net within the trabeculum ciliare (63,66).

The outer longitudinal portion of human CM connected with the scleral spur remains intact throughout life and does not show age-related degenerative change (65). There is little increase in intermuscular connective tissue and nearly no hyalinization of the interstitial tissue in this region. This longitudinal muscle portion may be important for keeping tension on the scleral spur to prevent collapse of Schlemm’s canal in old age (67). Studies in post-mortem human eyes suggest that the ageing human CM is still capable of moving forward in response to pilocarpine, albeit less so than in younger eyes (Lutjen-Drecoll, unpublished data), explaining why pilocarpine still enhances outflow facility in older subjects (68).

In rhesus monkeys under 20 years of age, intramuscular connective tissue is not increased, and spaces between anterior tips of the outer longitudinal CM remain free of pigment. After 20 years of age, the spaces between the muscle fiber bundles contain many more pigmented cells. After 24–25 years of age (equivalent to 70 human years), pigmented cells are present even between anterior longitudinal CM tips and the CM

loses its ability to move anteriorly-inwardly in response to pilocarpine although it retains the shape of a relaxed muscle. From age 26 to 35 years, the basement membranes of CM cells are thickened so that in some places the basement membranes of adjacent cells appear fused (69,70). The inability of the CM to move is attributed to agerelated changes in the posterior elastic tendons and their posterior attachment zones where collagen density is increased (61). CM fibers themselves show few changes with increasing age while extracellular material between the muscle bundles is slightly increased. In rhesus monkeys, an age-related decline in uveoscleral outflow is seen that is associated with thicker elastic fibers covering the anterior CM tips inserting into trabeculum ciliare and more ECM between the CM tips (20). The relatively immobile CM may also have less influence on the shape of the TM and outflow through that tissue. This could, in part, account for the age-related decline in the rhesus outflow facility in response to pilocarpine (71).

The non-pigmented ciliary epithelium shows an age-related increase in the number of mitochondria, cytoplasmic vacuoles, deposits of lipid granules, and fenestrations in its adjacent capillary endothelium. There are fewer membrane infoldings and associated ion pumps. The basal stroma is thickened (57,72–74). These findings may explain the decrease in aqueous humor formation seen with increasing age. An age-dependent loss of ciliary epithelial cells in humans has not been described (75).

Glaucomatous Changes in the Aqueous Outflow Pathways

There is more ECM material in the subendothelial region of Schlemm’s canal of POAG eyes than age-matched controls. Eyes with POAG have fine fibrillar material adhering to the cribriform elastic-like fibers that is not seen in controls. In POAG eyes, material associated with the thickened sheaths of elastic-like fibers predominates and may increase outflow resistance. Elastin gold-labeling shows increased localization to fine-fibrillar regions, with labeling density significantly greater in POAG than control eyes. The amount of TM sheath-derived plaques has been correlated with retinal nerve fiber loss in POAG, suggesting that some common factors underlie the formation of sheath-derived plaques and the optic neuropathy of glaucoma (47,51,76,77).

The trabeculectomy specimens of POAG patients show increased amounts of plaque material deposited within the cribriform layer and an abundance of long spacing collagen with the uveal meshwork showing paucity of cells. The cribriform layer often contains numerous enlarged cells with many small mitochondria and lysosomes but no prominent endoplasmic reticulum or Golgi complexes. More inner wall sheath-derived plaque material is seen in POAG than age-matched normal controls, and previous medical treatment does not seem to affect the amount of plaque material (78).

Schlemm’s canal is significantly smaller in POAG than control eyes. Inner wall pore density is about fivefold less in glaucoma compared with normal eyes. Were these findings to reflect true physiological differences, then they could contribute to the reduced outflow facility observed in POAG (79,80).

The amount of CM plaque material is significantly higher in POAG eyes than controls, and it does not correlate with age or inner wall plaque amounts, suggesting perhaps a multifactorial basis for plaque formation in these different regions. No differences are seen in CM shape and size, and amount of intermuscular connective tissue.

There are significant differences, however, in the amount of extracellular material at the anterior CM tips and their surrounding elastic fibers. In glaucomatous eyes, there is thickening of the sheaths surrounding the elastic-like fibers within the trabeculum ciliare (the extension of uveal meshwork over the ciliary muscle ligaments between the scleral spur and iris insertion) and also the anterior elastic tendons of the CM. The elastic-like fibers and their sheaths have a plaque-like appearance similar to that seen in the TM. In glaucomatous eyes, the anterior tendons of the CM and their fiber sheaths appear fused. Likewise, thickening of elastic-like fiber sheaths of the posterior connections of the CM to choroid and sclera and plate-formation are more prominent in glaucoma than age-matched control eyes (51,66,67). It is not known whether these changes influence uveoscleral outflow with age and glaucoma.

PUTATIVE MECHANISMS UNDERLYING AGEING

AND AGE-RELATED DISEASE

Gene Mutation

Insights into the basic biology of ageing have been gained from syndromes caused by single-gene mutations that have phenotypes of premature or accelerated ageing. Some syndromes are global, with many organ systems showing signs of accelerated ageing. A typical example is the rare autosomal recessive condition called Werner’s syndrome in which signs of accelerated ageing start after puberty as seen in skin changes, hair graying and thinning, cataracts (although not glaucoma), type 2 diabetes, and atherosclerosis, followed often by premature cancer, myocardial infarction, and premature death. Cultured cells derived from these people show a pattern of exaggerated replicative senescence, a phenomenon wherein cultured primary cells become less able to proliferate over time (see later) (http://www.pathology.washington.edu/research/Werner). Other syndromes are more limited in their phenotype and may primarily affect only one organ system such as dementias of the Alzheimer type (e.g., Familial Alzheimer’s, Parkinson’s, and Huntington’s) and low-density lipoprotein (LDL) mutations causing early-onset coronary artery disease (see Fig. 1).

Glaucoma arising by myocilin mutation is arguably an example of an ageing phenotype limited to one organ system. The myocilin protein is expressed at higher levels in the outflow pathways than any other ocular tissue (81,82). Myocilin gene mutations are found in some families with autosomal-dominant juvenile-onset openangle glaucoma (ADJOAG), and over 70 mutations have been reported (83–86). However, they are also seen in 3–4% of adult onset POAG so that the age-relatedness of myocilin glaucoma can be considered in two ways. It might primarily be a disease of younger people. Alternatively, it could represent a disease of older people appearing prematurely in a subset of young people. Adult-onset POAG is much more prevalent than ADJOAG. Adult-onset POAG is also more prevalent in older adults (1–2% in Caucasians over 40 years of age) than ADJOAG is in the juvenile age group (estimated 1:50,000). Even if only 3–4% of people with adult-onset POAG have myocilin glaucoma, we can still expect that there will be more adults with myocilin POAG than juveniles with myocilin ADJOAG. Statistically, this then makes myocilin

Environmental factors

Oxidative stress

Nutritional factors

Predisposition

DNA damage

Poor repair with age

Accumulation of mutations Cell senescence

Genomic instability

Ageing Age-related disease like glaucoma

Fig. 1. Hypothetical model of events leading to ageing and age-related disease.

glaucoma predominantly a disease of older people. The rare appearance of what is predominantly an aged phenotype in a juvenile population might then be thought of as a form of accelerated ageing.

Increasing evidence points to mutant forms of myocilin protein being poorly or not secreted from TM cells, with the protein instead accumulating in the endoplasmic reticulum (ER) (87–89). Wild-type myocilin is endoproteolytically processed near the C-terminus to yield a C-terminal fragment containing an olfactomedin-like domain and an N-terminal fragment containing a leucine zipper-like domain (90). Pathogenic mutations affecting endoproteolytic processing can cause myocilin to accumulate in the ER as insoluble aggregates. The amount of aggregation varies with the type of mutation and appears to be related to misfolding of the protein leading to TM cell morphological abnormality and death (91). The myocilin mutation, Pro370Leu, markedly inhibits endoproteolytic processing and is associated with particularly severe glaucoma, suggesting phenotypic correlation. Another myocilin mutation Tyr437His causes myocilin accumulation in the ER of lens epithelial cells, leading to nuclear cataracts and lens fiber rupture (92). Myocilin mutations can thus affect ER functioning wherein misfolded proteins accumulate and cause cell dysfunction and death (93,94), analogous to that seen in POAG and other ageing disorders. Even though myocilin mutations are a relatively uncommon cause of glaucoma, its study provides insights into mechanisms by which TM cells may die in the disease.

Oxidative Stress

The theory that aerobic metabolism and its generation of reactive oxygen species (ROS) contribute to ageing remains widely held 50 years after its proposal. Cells continuously make ROS, but they also have ROS-scavenging enzyme systems. Oxidative

stress occurs when the generation of ROS exceeds these anti-oxidant defenses. ROS can alter cellular lipids, proteins, and DNA such that continuous oxidative stress causes persistent cellular and genetic damage. There is evidence that mitochondrial function declines with age, with the mitochondria and anti-oxidant systems becoming more susceptible to oxidative damage (95). The well-known phenomenon of enhanced longevity from caloric intervention is believed to be due to reduced ROS generation causing less oxidative stress (96). The intracellular targets of ROS that presumably affect ageing and longevity are unknown.

Many intracellular compartments generate ROS, but the vast majority—about 90%—comes from mitochondria as a byproduct of oxidative phosphorylation. NADH or FADH oxidation generates an energy potential across the mitochondrial inner membrane that is used for phosphorylation. The electrons derived from NADH or FADH react with electron acceptors like oxygen and generate ROS.

The TM contains anti-oxidant enzymes such as superoxide dismutase, glutathione reductase, and catalase (97). The TM is in constant contact with the aqueous humor in which the key oxidant, hydrogen peroxide, is normally present as a result of reactions of ascorbic acid and trace metals (98,99). Additional hydrogen peroxide and ROS are generated by light-catalyzed reactions, metabolism, and phagocytosis or inflammation. Perfusing hydrogen peroxide in calf eyes reduces aqueous outflow if glutathione, an antioxidant, is absent from those eyes (100). Cultured human and monkey TM cells over-express alpha–beta crystalline, a heat shock protein, not only in response to heat shock but also to hydrogen peroxide exposure, reflecting cellular stress caused by oxidative injury (101). Human TM cells exposed to hydrogen peroxide in cell culture show reduced adhesion to the ECM proteins fibronectin, laminin, and collagen types I and IV. Actin and vimentin-containing structures are reorganized, and less paxillin and focal adhesion kinase is seen in focal contacts while the level of transcription factor NF-kB is enhanced (102). NF-kB is involved in regulating many aspects of cellular activity, including responses to stress and injury. Extensive and repeated oxidative stress in vivo may result in reduced TM cell adhesion and cell loss that affects the TM cell population and outflow regulation through this tissue.

The amount of DNA oxidative damage can be assessed by examining the levels of 8-hydroxy-2´-deoxyguanosine (8-OH-dG) in tissue. This nucleotide change is due to ROS reactions with DNA at C8 guanine residues which can lead to genetic changes such as point mutations. An age-related increase of 8-OH-dG is seen in post-mitotic tissues such as brain. TM obtained by trabeculectomy from eyes with POAG has almost five times the amount of 8-OH-dG compared with controls, indicating increased levels of oxidative DNA change in glaucomatous TM (103). Increased oxidative stress may lead to an over-expression of antioxidant enzymes, as seen in glaucoma eyes in which aqueous humor activities of the oxidant scavengers superoxide dismutase and glutathione peroxidase have been found to be elevated by 57% and threefold, respectively, compared with normal eyes (104). Conversely, the systemic levels of circulating glutathione have been found to be lower in individuals with glaucoma than age-matched controls (105). These findings raise the possibility of oxidative damage and impaired anti-oxidant defenses compromising cell function and survival in glaucoma.