Progress in Retinal and Eye Research 24 (2005) 612–637 Changes in aqueous humor dynamics with age and glaucoma B’Ann True Gabelt, Paul L. Kaufman Department of Ophthalmology and Visual Sciences, University of Wisconsin, F4/340 CSC, 600 Highland Avenue, Madison, WI 53792-3220, USA Abstract Changes in aqueous humor dynamics with age and in glaucoma have been studied for several decades. More recently, techniques have been developed which confirm earlier studies showing that outflow facility decreases with age and in glaucoma and add the newer finding that uveoscleral outflow also decreases. Morphologic studies in aging and glaucoma eyes have shown an increase in accumulation of extracellular material in both the trabecular meshwork and ciliary muscle and a loss of trabecular meshwork cells, which contribute to this reduction in outflow and result in an increase in intraocular pressure. A reduction in hyaluronic acid and increases in fibronectin and thrombospondin contribute to the change in the extracellular environment. Imbalances in responses to age-related stresses such as oxidative damage to long-lived molecules, protein cross-linking and loss of elasticity could trigger excess production of factors such as transforming growth factor b, interleukin-1 and CD44S that could stimulate pathways leading to increases in fibronectin, transformation of trabecular meshwork cells to a myoepithelial state and decrease the breakdown in extracellular matrix material, allowing excess to accumulate. Ultimately trabecular outflow and uveoscleral outflow are reduced and intraocular pressure becomes elevated, adding more stress and perpetuating the pathological condition. Future research to identify additional factors and clarify their roles in these processes could lead to alternative therapies for age and glaucoma related changes in the eye. r 2005 Elsevier Ltd. All rights reserved. ARTICLE IN PRESS www.elsevier.com/locate/prer Contents 1. Introduction ............................................................................. 613 2. Aqueous humor dynamics ................................................................... 614 2.1. Intraocular pressure (IOP) .............................................................. 614 2.2. Aqueous humor outflow ................................................................ 615 2.2.1. Trabecular outflow .............................................................. 615 2.2.2. Uveoscleral outflow (Fu) ......................................................... 616 2.3. Aqueous humor formation (AHF) ......................................................... 616 2.4. Episcleral venous pressure ............................................................... 617 2.5. Ocular rigidity ....................................................................... 617 2.6. Iris permeability ...................................................................... 617 2.7. Aqueous humor turnover rate ............................................................ 617 3. Morphology and extracellular material or matrix (ECM) ............................................. 617 3.1. Trabecular meshwork (TM) and Schlemm’s canal .............................................. 617 3.2. Ciliary muscle (CM) ................................................................... 620 3.3. Ciliary epithelium..................................................................... 623 1350-9462/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.preteyeres.2004.10.003 Corresponding author. Tel.: +1 608 263 6074; fax: +1 608 263 1466. E-mail address: [email protected] (P.L. Kaufman).
Transcript
ARTICLE IN PRESS
Contents
1. Introduc
2. Aqueous
2.1. In
2.2. Aq
2.2
2.2
2.3. Aq
2.4. Ep
2.5. Oc
2.6. Iri
2.7. Aq
3. Morphol
3.1. Tr
3.2. Ci
3.3. Ci
1350-9462/$ - se
doi:10.1016/j.pr
�CorrespondE-mail addr
Progress in Retinal and Eye Research 24 (2005) 612–637
www.elsevier.com/locate/prer
Changes in aqueous humor dynamics with age and glaucoma
B’Ann True Gabelt, Paul L. Kaufman�
Department of Ophthalmology and Visual Sciences, University of Wisconsin, F4/340 CSC, 600 Highland Avenue, Madison, WI 53792-3220, USA
Abstract
Changes in aqueous humor dynamics with age and in glaucoma have been studied for several decades. More recently, techniques
have been developed which confirm earlier studies showing that outflow facility decreases with age and in glaucoma and add the
newer finding that uveoscleral outflow also decreases. Morphologic studies in aging and glaucoma eyes have shown an increase in
accumulation of extracellular material in both the trabecular meshwork and ciliary muscle and a loss of trabecular meshwork cells,
which contribute to this reduction in outflow and result in an increase in intraocular pressure. A reduction in hyaluronic acid and
increases in fibronectin and thrombospondin contribute to the change in the extracellular environment. Imbalances in responses to
age-related stresses such as oxidative damage to long-lived molecules, protein cross-linking and loss of elasticity could trigger excess
production of factors such as transforming growth factor b, interleukin-1 and CD44S that could stimulate pathways leading toincreases in fibronectin, transformation of trabecular meshwork cells to a myoepithelial state and decrease the breakdown in
extracellular matrix material, allowing excess to accumulate. Ultimately trabecular outflow and uveoscleral outflow are reduced and
intraocular pressure becomes elevated, adding more stress and perpetuating the pathological condition. Future research to identify
additional factors and clarify their roles in these processes could lead to alternative therapies for age and glaucoma related changes
B.T. Gabelt, P.L. Kaufman / Progress in Retinal and Eye Research 24 (2005) 612–637 613
1. Introduction
Primary open angle glaucoma (POAG) is an age-related disease (reviewed in (Friedman et al., 2004;Quigley and Vitale, 1997)) (Fig. 1). Therefore it is likelythat variations or excesses in the normal aging processescould shift the balance toward the initiation ofglaucoma and its progression. Relevant to aqueoushumor dynamics, these processes result in accumulationof extracellular material in the trabecular (Gottanka etal., 1997; Lutjen-Drecoll et al., 1986b; Rohen andWitmer, 1972) and uveoscleral outflow (Fu) (Lutjen-Drecoll, 1998b; Lutjen-Drecoll et al., 1986a) pathwaysleading to an elevation in intraocular pressure (IOP)possibly initiating a vicious cycle leading to furtherreductions in outflow and IOP elevation. Severaltheories have been put forth to postulate what types ofaging processes are most relevant for triggering theonset of glaucoma.One hypothesis is that aging processes in the eye occur
as a consequence of degradation of enzymes thatnormally metabolize and detoxify hydrogen peroxideand other free radicals. Hydrogen peroxide and freeradicals induce irreversible deleterious effects ondifferent eye tissues. In the trabecular meshwork(TM), modification of the glycosaminoglycan secretorypatterns can occur which could lead to outflowobstruction and IOP elevation. These processes maybe exacerbated during inflammation when oxidationproducts increase (Green, 1995). It appears that thelevels of oxidation materials such as hydrogen peroxidedo not themselves vary with age, but rather there is adecrease with age of the protective mechanisms (Green,1995).Cells respond to mechanical stress by altering their
behavior in a variety of ways. These responses addressthe immediate need for protection and also set in motion
both short and long-term programs for mediatingadaptive tissue remodeling. Extensive and repeatedoxidative stress in vivo may induce reduction of TMcell adhesion resulting in cell loss, compromised TMintegrity, and pathologic consequences (Zhou et al.,1999a, b).Studies of diseases in many organ systems tell us that
the gene products induced as part of stress responseshave much potential to cause deleterious effects ontissues when expressed on a chronic basis. Diseases ofthe vasculature provide an excellent example because ofthe structural and functional similarities to the aqueousoutflow pathways. Endothelial leukocyte adhesionmolecule-1 (ELAM-1) is the earliest marker of theendothelial response to sub-lethal injury that occurs invascular disease (Gimbrone et al., 1997; Price andLoscalzo, 1999). ELAM-1 was found to be consistentlypresent on TM cells in the outflow pathways of eyes withglaucomas of diverse etiologies (Wang et al., 2001).Expression of ELAM-1 was controlled by activation ofan interleukin-1 (IL-1) autocrine feedback loop throughtranscription factor NF-kB, and activity of this signal-ing pathway was shown to protect TM cells againstoxidative stress. However, chronic stress and activationof these mechanisms may contribute to the pathophy-siology of glaucoma and vascular diseases (Wang et al.,2001).Protein cross-linking and loss of elasticity could
change the dynamics of the interactions betweenthe ciliary muscle (CM) and TM due to theintimate anatomic relationship between them. CMimmobility could adversely affect aqueous outflow.The rapid macro- and micro-contractions andrelaxations of the muscle that occur during normalvisual activity may generate tremendous distortionalforces within the TM. In conjunction with aqueousflow, this could help cleanse the meshwork of
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Fig. 1. Prevalence of glaucoma in white (A) and black and Hispanic
facility; Cton ¼ tonographic outflow facility; Fa ¼ aqueous flow;
Fu ¼ uveoscleral outflow; IOP ¼ intraocular pressure; K ¼ corneal
thickness; Pev ¼ episcleral venous pressure.aComparing the two groups with unpaired two-tailed t-test. From
Toris et al. (1999) with permission.
B.T. Gabelt, P.L. Kaufman / Progress in Retinal and Eye Research 24 (2005) 612–637614
debris. When the aging CM loses its ability tomove, periodic deformation of the TM would bereduced or cease and extracellular material couldaccumulate within its interstices, in turn producingincreased outflow resistance and elevated IOP (Kauf-man and Gabelt, 1995). We will review the changes inaqueous humor dynamics and anterior segment mor-phology that have been reported to take place in agingand glaucoma. We will also review changes in metabo-lism and aqueous humor factors that may influence theonset and progression of the observed changes andwhether they support existing or alternative theories ofglaucoma.We will limit the scope of this chapter primarily to
human and nonhuman primate studies. Human glauco-ma data will be limited mainly to POAG.
2. Aqueous humor dynamics
2.1. Intraocular pressure (IOP)
Aging: IOP in normal healthy humans remainsrelatively stable or slightly increases with age in manyWestern populations (Armaly, 1967; Becker, 1958;Brubaker et al., 1981; Gaasterland et al., 1973). A morerecent study of aqueous humor dynamics in healthysubjects 20–30 or460 years of age, also showed no age-related IOP increase (Toris et al., 1999) (Table 1).Conversely, a study done in a Japanese populationfound that IOP decreases with age. Differences wereattributed to blood pressure, obesity and other cardiacrisk variables (Nomura et al., 2002; Shiose, 1990). Largedemographic studies in humans in the United Statesincluding all eye conditions found an increase in IOPwith age (Hiller et al., 1982; Klein et al., 1992).Similarly, IOP in normal, healthy, free-ranging rhesus
monkeys remains relatively constant with age, after aninitial juvenile hypertensive phase (Bito et al., 1979;DeRousseau and Bito, 1981; Kaufman and Bito, 1982).More recently, a study of cage-housed rhesus monkeysages 3–29 years, which is the human equivalent ofapproximately 8–73 years, found a slight increase in IOPwith age (Gabelt et al., 2003) (Table 2).
Glaucoma: Elevated IOP is the greatest risk factor forglaucoma (Armaly et al., 1980; Leske, 1983; Leske et al.,2003; Sommer, 1996), the incidence of which increaseswith age (Armaly et al., 1980; Kini et al., 1978; Leske,1983; Leske et al., 2003) (Fig. 2). However no specificIOP can differentiate ‘‘normal’’ eyes from those that willdevelop glaucomatous damage although 21mmHg is
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Table 2
Aging effects on aqueous formation and drainage in Rhesus monkeys
3–10 yr; n ¼ 11 19–23 yr; n ¼ 8 25–29 yr; n ¼ 5 3–23 yr; n ¼ 19
Data are mean7s.e.m.: IOP ¼ intraocular pressure; AC ¼ anterior chamber; AHF ¼ aqueous humor formation; Fu ¼ uveoscleral outflow;
FTB ¼ flow to blood. Results in the 25- to 29-year age group were significantly different from those in the 3- to 10-, 19- to 23-, and 3- to 23-year age
groups, according to the unpaired t-test for differences with unequal variances compared to 0.0: �po0:05: (From Gabelt et al. (2003) with
permission.)an ¼ 10:bn ¼ 7:cn ¼ 17:
Fig. 2. Relative risk of POAG at different levels of screening IOP
among subjects studied in the Baltimore Eye Survey. From Sommer
(1996) with permission.
Fig. 3. A. Outflow facility vs age in 30 human subjects. From Croft et
al. (1996) with permission. B. Outflow facility vs age in 17 rhesus
monkeys. From Gabelt et al. (1991) with permission. Lines represent
least-squares regression of ordinate variable on abscissa variable. r,
correlation coefficient; P, probability that the slope is different from
0.0 by the two-tailed paired t-test.
B.T. Gabelt, P.L. Kaufman / Progress in Retinal and Eye Research 24 (2005) 612–637 615
considered the demarcation for ocular hypertension(reviewed in Schottenstein, 1996). Treating ocularhypertension with glaucoma medication is efficaciousin delaying or preventing the onset of glaucoma (Kass etal., 2002; Lee et al., 2003). The magnitude of the initialIOP and its reduction with therapy are major factorsinfluencing glaucoma progression (Leske et al., 2003).
2.2. Aqueous humor outflow
2.2.1. Trabecular outflow
Aging: Studies in nonglaucomatous humans demon-strated that total outflow facility decreases with age.Measurements were made in vivo tonographically or byperfusion after enucleation (Becker, 1958; Gaasterlandet al., 1978). Tonographic studies also showed age-related decreases in trabecular facility and pseudofacilityas well as total facility (Gaasterland et al., 1978; Kupfer,1973). The age-related decrease in facility was about30% from the youngest (o40 years) to the oldest (460
years) (Becker, 1958; Gaasterland et al., 1978). Morerecent tonographic studies yielded similar results (Croftet al., 1996; Toris et al., 1999) (Fig. 3a). Toris’ data inhumans shows a slight but insignificant decrease inoutflow facility from 0.25 ml/min/mmHg in 20–30-yearsold to 0.19 ml/min/mmHg in those over 60 years (Toris
ARTICLE IN PRESSB.T. Gabelt, P.L. Kaufman / Progress in Retinal and Eye Research 24 (2005) 612–637616
et al., 1999). However, the age-related decrease intonographic outflow facility could be explained by thedecrease in pseudofacility alone (Toris et al., 1999)which could, in part, be due to the increase in ocularrigidity (see below) with age. Fluorophotometric mea-sures of outflow facility, which are not affected bypseudofacility and ocular rigidity (Toris et al., 1995),showed no differences with age (Toris et al., 1999)(Table 1).In rhesus monkeys, baseline outflow facility measured
by two-level constant pressure perfusion (Barany, 1964)decreased with age (Gabelt et al., 1991) (Fig. 3b). Theinability of atropine to completely eliminate the age-related facility decline in monkeys indicated the presenceof atropine-independent, facility-relevant age-dependentchanges in the TM itself, such as loss of cells or build-upof extracellular material (Kiland et al., 1997).
Glaucoma: In primary open angle glaucoma patientsthere was an age-related facility decrease similar inmagnitude to that reported for non-glaucomatouspatients (Becker, 1958). A longitudinal study in un-treated ocular hypertensives demonstrated a progressivefacility decrease over a 10-year period (Linner, 1976).The absolute value of outflow facility in open-angleglaucoma patients was significantly less than in age-matched controls (Larsson et al., 1995). In ocularhypertensives, fluorophotometric outflow facility wassignificantly decreased compared to age-matched con-trols (Toris et al., 2002) (Table 3).
2.2.2. Uveoscleral outflow (Fu)
Aging: In humans, early studies of Fu conducted inelderly humans with posterior segment tumors indicatedthat Fu accounts for no more than 25% of total aqueousoutflow. In young healthy humans, Fu was calculated toaccount for 36% of total outflow but accuracy was inquestion with tonography measures taken at low IOPs(Townsend et al., 1980). More recently, Fu calculatedfrom fluorophotometric measures of outflow facility and
Table 3
Average of both eyes of patients with ocular hypertension or age-matched o
venous pressure. From Toris et al. (2002) with permission.
aqueous humor formation (AHF) was determined toaccount for 54% of total aqueous drainage in younghealthy humans compared to 46% in older healthysubjects (Toris et al., 1999) (Table 1).Uveoscleral drainage, measured by isotope accumula-
tion techniques in young healthy monkey eyes, ac-counted for 45–70% of total aqueous drainage (Bill,1971). In rhesus monkeys Fu significantly decreased byabout half in 25–29-year olds compared to 3–10-yearolds (Gabelt et al., 2003) (Table 2).
Glaucoma: Fu calculated from fluorophotometricmeasures of AHF and outflow facility comprised 78%of the total aqueous drainage in patients with POAG,although the absolute values were lower than for healthysubjects (Yablonski et al., 1985). However, a roughestimate from Toris’ data suggests Fu makes up only25% of AHF in ocular hypertensives compared 42% innormotensive controls (Toris et al., 2002) (Table 3).
2.3. Aqueous humor formation (AHF)
Aging: Over the years, several studies have beenconducted on the effect of aging on the rate of AHF.Tonographic (Becker, 1958; Gaasterland et al., 1978;Kupfer, 1973) and fluorophotometric (Bloom et al.,1976; Brubaker et al., 1981; Diestelhorst and Kriegl-stein, 1992) studies in humans consistently demonstratean age-related decline in the rate of aqueous productionwhich amounts to �15–35% over the 20–80 year agespectrum (Becker, 1958; Bloom et al., 1976; Brubaker etal., 1981; Gaasterland et al., 1978; Kupfer, 1973; Toris etal., 1999) (Table 1). The decline probably occursthroughout adult life and is estimated to be0.003–0.015 ml/min/year or 2.4%/decade (Becker, 1958;Brubaker et al., 1981; Kaufman, 1987; Toris et al.,2002). In some studies, the decline became moreprecipitous after the age of 60, and was estimated tobe 1–2%/year or 0.025 ml/min/year (Becker, 1958;Brubaker et al., 1981). The reduction in AHF with age
cular normotensive volunteers
Ocular normotension
N Mean7SEM p a
55 14.970.3 o0.000155 9.070.2 0.1
55 2.5870.10 0.8
54 1.0970.11 0.005
54 0.2770.02 o0.000155 19877 0.8
55 52575 0.2
ng an unpaired, two-tailed t-test. ACvol, anterior chamber volume; K,
ARTICLE IN PRESSB.T. Gabelt, P.L. Kaufman / Progress in Retinal and Eye Research 24 (2005) 612–637 617
counteracts the resistance to outflow with age so that theIOP normally does not become substantially elevatedwith age.The ultrafiltration component of AHF is pressure
sensitive and decreases with increasing IOP. Thispressure-sensitive decrease in inflow, termed pseudofa-cility, mimics an increase in outflow as measured withtonography and constant pressure perfusion. An age-dependent decline in pseudofacility of 33% fromyounger (20–40 years) to older (50–80+ years) subjectswas demonstrated (Gaasterland et al., 1978; Kupfer,1973).In a smaller sample of rhesus monkeys, AHF was
unchanged with age (Gabelt et al., 2003) (Table 2).Glaucoma: The rate of AHF measured by fluoropho-
tometry was no different during the day in 20 patientswith open-angle glaucoma who had discontinued theirtherapy for 6 weeks compared to their age-matchedcontrols. A significantly higher rate of AHF wasmeasured in the glaucoma patients compared to controlsat night (Larsson et al., 1995).The rate of AHF is no different in ocular hyperten-
sives than in age-matched controls. In both groups AHFdecreases but not significantly with age (Toris et al.,2002) (Table 3).
2.4. Episcleral venous pressure
Aging and glaucoma: Episcleral venous pressure doesnot appear to change with age in humans (Gaasterlandet al., 1978; Kupfer, 1973; Toris et al., 1999; Zeimer etal., 1983).In ocular hypertensive patients, there is a nonsignifi-
cant increase in episcleral venous pressure compared tonormal volunteers (Toris et al., 2002) (Table 3).
2.5. Ocular rigidity
Aging: Ocular rigidity determined from paired in-dentation readings using two different weights on theSchiotz tonometer increases by about 25% in oldercompared to younger subjects (Armaly, 1959; Gaaster-land et al., 1978).
2.6. Iris permeability
Aging: Iris permeability to intravenous fluoresceintends to increase by about 13%/decade between the agesof 20 and 80 years, although this is not statisticallysignificant (Brubaker et al., 1981).
2.7. Aqueous humor turnover rate
Aging: Although total aqueous flow decreases withage, there is actually a small (�5%/decade) butsignificant increase in the turnover rate of anterior
chamber aqueous with age (Brubaker et al., 1981). Thismay in part be due to the decrease in anterior chamberdepth and volume with age (Bito et al., 1982) as a resultof increased lenticular volume (Bito et al., 1982).
3. Morphology and extracellular material or matrix
(ECM)
3.1. Trabecular meshwork (TM) and Schlemm’s canal
Aging: A progressive decrease in human TM cellcounts with age at a rate of 0.58–0.85%/year wasreported (Alvarado et al., 1981; Grierson and Howes,1987; Grierson et al., 1982). In addition there was anincrease in pigmentation of TM cells, an increasedincidence of cells detaching from trabeculae and aprogressively greater incidence of fusion betweenadjacent trabeculae as the outflow system ages (Griersonet al., 1982). At 20 years of age the TM population is inthe order of 1,200,000 cells, decreasing to �500,000 cellsby 80 years, indicating a loss of about 12,000 cells/year.Possible explanations for this loss are that TM cells invivo have only a limited capacity for replication. TMcells are avid phagocytes (Rohen and Van der Zypen,1968; Sherwood and Richardson, 1988; Zhou et al.,1996), have the capacity to migrate (Calthorpe andGrierson, 1990; Zhou et al., 1999a, b), produce ECMelements (Yue, 1996) and transduce signals uponattachment to the ECM (Zhou et al., 2000). As TMcells become more committed to phagocytosis with agein order to remove increasing quantities of debris, theymay eventually detach from the trabeculae and be lostvia Schlemm’s canal. Decreased TM cellularity with agecould alter the synthetic and catabolic control of theextracellular environment (Grierson et al., 1982). Alter-natively, the accumulation of debris could be toxic toTM cells or sequester them from the aqueous humornecessary to supply them with nutrients and removetoxic metabolites (Kaufman and Gabelt, 1995).TM cell numbers in the cynomolgus monkey mesh-
work continuously decreases with increasing agealthough the degree of this decline differs within thevarious decades (Rohen et al., 1993). In the TM ofaging rhesus monkeys, the number of TM cells alsodecreases, and an increase in fibrillar material andsheath-derived plaques is observed (Gabelt et al., 2003)(Fig. 4A) consistent with the decrease in outflowfacility reported in both humans and monkeys (Croftet al., 1996; Gabelt et al., 1991; Kiland et al., 1997; Toriset al., 1999).The cribriform plexus (network of elastic-like fibers in
the cribriform area) connects with the endothelium ofSchlemm’s canal by fine radially oriented fibrils.Anterior CM tendons are anchored within this plexusso that contraction of the muscle may influence the
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Fig. 4. (A) (left) High magnification of sheath derived plaques (arrows) underneath the inner wall of Schlemm’s canal (SC) in a 34-year-old monkey.
EL, elastic fiber. Scale bar, 1 mm. From Gabelt et al. (2003) with permission. (B) (right) Electron micrograph of the cribriform layer in a case ofchronic simple glaucoma to demonstrate the sheath-derived (SD) plaque material (arrows). Asterisk ¼ subendothelial granular material. The SD
plaques are mainly formed by the cribriform elastic-like (El) network with its sheaths and connecting fibrils (C) with their surrounding sheath
material (sagittal section, trabeculectomy specimen). E ¼ endothelial lining of Schlemm’s canal; SC ¼ lumen of Schlemm’s canal. From Lutjen-
Drecoll et al. (1986b) with permission.
Fig. 5. Schematic drawing of the cribriform elastic-like plexus (CP)
connected to the inner wall endothelium (E) of Schlemm’s canal by
connecting fibrils (CF). The outer tendons of the CM, insert into the
CP so that muscle contraction can influence the aqueous humor
pathways through the cribriform region and the giant vacuoles of the
endothelium (both black) into Schlemm’s canal. Arrow indicates
footlike connection between endothelial and subendothelial trabecular
cell. From Lutjen-Drecoll (1998a) with permission.
B.T. Gabelt, P.L. Kaufman / Progress in Retinal and Eye Research 24 (2005) 612–637618
width of the cribriform layer and change the form of theintercellular spaces of that area, thereby influencingoutflow resistance (Fig. 5) (Lutjen-Drecoll and Rohen,1984; Rohen, 1982, 1983). The elastic-like fibers of thecribriform plexus are surrounded by a sheath consistingof fine fibrils embedded in a more or less homogeneousmatrix, rich in chondroitin sulfates (Lutjen-Drecoll etal., 1981). In older eyes (50–80 years), the elastic-likefibers in the cribriform meshwork become thicker andcoarser, primarily because of an age-related increase inthe amount of sheath material (Lutjen-Drecoll et al.,1986b; Rohen et al., 1981) (Fig. 6). One theory is that aslong as the meshwork remains large enough and retainsits ability to expand in response to CM tone, there islittle effect of the accumulating sheath material onoutflow resistance (Rohen et al., 1981). However, astrong correlation exists between the amount of sheath-derived (SD) plaques formed by trabecular cells and theextent of retinal nerve fiber loss in the posterior eyesegment in POAG. No significant correlation was foundbetween IOP and nerve fiber counts. This suggested thatfactors influencing the formation of sheath-derivedplaques also play an important role in nerve fiber lossbefore or in conjunction with IOP elevation (Gottankaet al., 1997). Nonetheless, studies have shown that IOPdoes correlate with the risk of developing glaucoma(Kass et al., 2002) and with the risk of progression inestablished POAG (AGIS, 2000; Collaborative Normal-Tension Glaucoma Study Group (CNTG), 2000; Leskeet al., 2003).A study of the composition of ECM materials in the
cribriform TM and inner wall of Schlemm’s canal ofnormal human eyes indicates that the amorphousbasement-membrane-like materials are made up ofmajor structural proteins including collagen type IV,laminin, and fibronectin (Hann et al., 2001; Marshall etal., 1990, 1991; Ueda et al., 2002). mRNA for all threeproteins is present in most trabecular cells throughout
the meshwork (Hann et al., 2001). Elastin localizes tothe central core of SD plaques (Gong et al., 1989; Uedaet al., 2002). Microfibrillar associated glycoprotein(MAGP)-1 and fibronectin, along with fibrillin-1,localize mostly within the peripheral mantle of thesheath surrounding the elastin core in SD plaques of thecribriform TM. The fibrillin-containing microfibrillarsystem in normal ocular tissues was suggested to have asubstantial role in the maintenance of tissue integrity byproviding tensile strength and flexibility required duringregulation of aqueous humor outflow (Schlotzer-Schre-bardt et al., 1997; Ueda et al., 2002). Myocillin localizesextracellularly in the cribriform TM to the microfibrillararchitecture of SD plaques. It is unknown whethermyocilin is an integral constituent of the microfibrils ormerely forms an association with microfibril-associatedcomponents. The microfibril-associated elements within
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Fig. 6. Scatter diagrams showing the relationship between inner wall (A, B) and outer wall (C, D) plaques with age in normal eyes (A, C) and in
POAG eyes (B, D). There is a significant correlation between inner and outer wall plaques in normal eyes but not in POAG eyes. From Lutjen-
Drecoll et al. (1986b) with permission. (A) r ¼ 0:49; po0:01; (B) r ¼ 0:18; (C) r ¼ 0:57; po0:01; (D) r ¼ �0:17:
B.T. Gabelt, P.L. Kaufman / Progress in Retinal and Eye Research 24 (2005) 612–637 619
the banded materials in the sheath of SD plaquescorresponds to the region where pathologic changes areoften observed in the eyes of patients with POAG (Uedaet al., 2002). Similar localization patterns for myocilinwere demonstrated in the corneoscleral meshwork(Ueda and Yue, 2003). In HTM cells in vitro treatedwith dexamethasone or dexamethasone+ascorbate,myocillin colocalizes with fibronectin, laminin, and typeIV collagen (Filla et al., 2002).Extracellular myocillin also elicites anti-adhesive and
counter migratory effects on TM cells (Wentz-Hunter etal., 2004). The heparin II domain of fibronectin inparticular was found to be the interacting site formyocilin (Filla et al., 2002). The heparin II domain,where fibronectin is linked to the actin cytoskeleton,lends mechanical stability and transduces signals fromthe exterior to the interior of the cells (Arnaout et al.,2002; Woods et al., 1986). Inhibition of cell attachmentsby myocilin altered the actin cytoskeletal structure andfocal contact formation (Wentz-Hunter et al., 2004).Under conditions of excessive phagocytosis, inflamma-tion and injury, TM cells migrate through Schlemm’scanal to clear the angle (Alvarado and Murphy, 1992;Grierson and Hogg, 1995). The anti-adhesive and anti-
migratory effects of myocilin on TM cells were notobserved with corneal fibroblasts (Wentz-Hunter et al.,2004) in keeping with the notion that myocilin distribu-tion and regulation may be TM cell specific (Polansky etal., 2000, 1989; Wentz-Hunter et al., 2003).In younger eyes, a-smooth muscle(sm) actin filaments
are seen in nearly all cells of the TM. If these cells arecontractile, they could influence outflow facility byconfigurational alterations of the outflow pathway.With increasing age, fewer cells stain for a-sm actinand are localized mainly in the posterior part of the TMand in the scleral spur. No differences are found betweenglaucomatous eyes and normal eyes of the same agegroup. Concomitant with the loss of a-sm actinfilaments in the TM with age, an increase of synthesizingorganelles such as rough endoplasmic reticulum wasobserved which could contribute to the age-relatedincrease of extracellular material in this region. Both theincrease in synthetic activity and loss of contractileprotein might contribute to a decrease in outflow facilitywith age which is more pronounced with glaucoma(Flugel et al., 1992). Substances which contract isolatedTM decreased outflow rate in isolated perfused anteriorsegments with intact TM but without CM, suggesting a
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functional antagonism between the TM and CM(reviewed in Wiederholt, 1998). Stretching of TM causesalterations in the cytoskeletal network and signalingcascades (Tumminia et al., 1998). Other isoforms ofactin are observed in all cellular constituents of theoutflow pathway (de Kater et al., 1992). Protein profilesfrom the TM of human eyes of various ages show adecrease in G-actin with age as well as an increase ofcomponents of type IV collagen (Millard et al., 1987).Age-related changes in the actin cytoskeleton werereported in other cell types (Galustian et al., 1995;Labuhn and Brack, 1997; Reed et al., 2001). Furthercharacterization of the age and glaucoma relatedchanges in the cytoskeleton of the TM are needed.
TM: glaucoma: Quantitative morphological studiesrevealed a significant increase in extracellular material inthe subendothelial region of Schlemm’s canal inglaucomatous eyes compared to age-matched normalcontrols. In eyes with POAG, material derived from (oradhering to) the thickened sheath of the elastic-likefibers predominates and presumably contributes to theincrease in outflow resistance (Gottanka et al., 1997;Lutjen-Drecoll et al., 1986b; Rohen and Witmer, 1972)(Fig. 4B). As stated previously, factors influencing theformation of sheath-derived plaques may also contri-bute to nerve fiber loss before or in conjunction withIOP elevation (Gottanka et al., 1997).In an ultrahistochemical study using enzyme digestion
methods, eyes with chronic simple glaucoma had anadditional fine fibrillar material adhering to the cribri-form elastic-like fibers or the connecting fibrils, whichwere not present in normal eyes (Lutjen-Drecoll et al.,1981).In POAG, the amount of inner-wall plaques tend to
be greater than that of the outer wall. If factors in theaqueous can promote plaque formation by endothelialcells of the outflow tissues, the trabecular cells would bemore exposed to these factors than would outer-wallcells. If material deriving from other areas in the eyeflows into the meshwork and becomes incorporated intothe elastic-like fiber sheaths, these substances would alsoreach the cribriform plexus more easily than the plexusin the outer wall. Quantitation of plaque material didnot permit distinction between the normal and glauco-matous state in the individual case since there wasconsiderable overlap between the groups (Lutjen-Dre-coll et al., 1986b) (Fig. 6).Thirty-one trabeculectomy specimens of patients
suffering from POAG were ultrastructurally and quan-titatively analyzed. Most specimens revealed thickenedtrabeculae, increased amounts of plaque-material de-posited within the cribriform layer and an abundance oflong spacing (lattice) collagen. The uveal meshwork waspartly deprived of cells. The cribriform layer oftencontained numerous enlarged, light cells with manysmall mitochondria and lysosomes but no prominent
endoplasmic reticulum or Golgi complexes. Higheramounts of SD plaque material were present in theinner wall than normal controls of a similar age range.There was no difference in the amount of SD plaquematerial in eyes that received medical treatmentcompared to those who did not (Rohen et al., 1993).In all examined cases of POAG, the amount of
positively labeled gold particles for elastin within thearea containing fine fibrillar-like material that waspresent in the subendothelial layer of Schlemm’s canalwas distinctly larger than in age matched normal eyes.Labeling for elastin within the elastic-like fibers wassimilar to that observed in normal eyes (Umihara et al.,1994). Elastin particle density human TM specimens was6.372.1/mm2 in glaucoma eyes and 0.770.3/mm2 incontrol eyes (Umihara et al., 1994).In glaucomatous eyes, the decreased thickness of the
cribriform meshwork and the shortening of the con-necting fibrils could reduce the ability of the tissuebetween the cribriform plexus and the inner wallendothelium to expand and would diminish the influ-ence of CM tone on outflow resistance. This might resultin an underperfusion of the area concerned, which inturn would lead to an even greater increase in theamount of extracellular material and in rigidity of thetissue. Thus a vicious cycle could develop, which mightbe one factor in the pathogenesis of glaucoma (Rohenet al., 1981).A more extensive review of functional TM morphol-
ogy in primate eyes may be found in Lutjen-Drecoll(1998a).The dimensions of Schlemm’s canal in glaucomatous
human eyes are significantly smaller than those innormal eyes. This reduction in Schlemm’s canal dimen-sions accounts for approximately half of the decrease inoutflow facility observed in POAG eyes (Allingham etal., 1996). Inner wall pore density is reduced byapproximately five-fold in glaucomatous eyes comparedto normal eyes. If pores are physiological structures, theelevated IOP in glaucoma may be associated withdecreased porosity of the inner wall endothelium.However the possibility that inner wall pores arefixation-induced artifacts cannot be excluded (Johnsonet al., 2002).Myocilin protein is the product of a gene that has
been linked directly to both juvenile- and adult-onsetopen angle glaucoma (Alward et al., 1998; Stone et al.,1997). Excess secretion of myocilin into the extracellularenvironment of the glaucomatous TM could contributeto the loss of TM cell adhesiveness as described above(Wentz-Hunter et al., 2004).
3.2. Ciliary muscle (CM)
CM: aging: CM contraction in response to peripheralcholinergic (reviewed in Kaufman and Gabelt, 1992) or
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central electrical (James et al., 2004) stimulationdramatically increases aqueous humor outflow byaltering the morphology of the TM and Schlemm’scanal (Lutjen, 1966) (Fig. 7) as referred to previously. Inprimate eyes, the posterior tendons of the CM insertinto Bruch’s membrane and into the elastic network ofthe choroid (Tamm et al., 1991). The collagen fibers ofthe choroid and sclera thicken with increasing age(Lutjen-Drecoll et al., 1982). The elastic fibers of thechoroid, Bruch’s membrane and around the optic nervehead lost their elastin core and appear more electrondense with age. The elastic-like fibers of the suprachor-oidal layer and sclera undergo changes with ageresembling those seen in the TM and CM tips becomingthicker and containing more cross-linked microfibers.Thickened sheaths of elastic-like fibers often merge inthe suprachoroidal layer and sclera forming dense platesof cross-linked fibrillar material (Lutjen-Drecoll,1998b).
Fig. 7. Histological sagittal sections through the CM of monkey eyes
treated with atropine (top) and pilocarpine (bottom) (� 24.8). During
contraction the CM moves anterior inwardly, resulting in a spreading
of the trabecular meshwork lamellae and widening of Schlemm’s canal.
From Lutjen (1966) with permission.
The shape of the CM is very different in old rhesusmonkey eyes versus old human eyes. In the elderlyhuman, the CM is short and forms a prominent inneredge of circular muscle fibers (Tamm et al., 1992a, b).One theory is that the posterior elastic-like tendons ofthe human eye lose their elasticity with age so the musclecannot be pulled backwards during disaccommodation,retaining its contracted appearance. Also, there are fewpigmented cells in the connective tissue spaces of elderlyhuman eyes (Fig. 8) (Lutjen-Drecoll, 1998b).Age-related changes in the morphology of the human
CM are significantly different from those of rhesus andother monkeys eyes (Lutjen-Drecoll et al., 1988a, b;Tamm et al., 1992a, b). In humans, the extracellularmaterial between the muscle bundles significantlyincrease with age, and are especially prominent in theintermediate reticular portion of the muscle facing theanterior chamber (Tamm et al., 1992a, b). At the CMtips, the elastic-like tendons and elastic-like fibers withinthe trabeculum ciliare undergo age-related changessimilar to those described in the TM. The sheaths ofthe elastic-like fibers thicken markedly. Broad plates ofbanded collagen are seen where the tendon sheathsconnected to the sheaths of the elastic-like net within thetrabeculum ciliare. The size of the open connectionsbetween the anterior chamber and CM decrease (Lutjen-Drecoll, 1998b). In normal eyes the amount of the CMplaques increase with age and correlate with the amountof plaques in the inner wall of Schlemm’s canal (Lutjen-Drecoll et al., 1986a).In humans, the outer longitudinal portion of the CM
which connected with the scleral spur does not showgreater degenerative changes with increasing age. Thismuscle portion remains intact throughout life. There islittle increase in intermuscular connective tissue andnearly no hyalinization of the interstitial tissue in thisregion (Tamm et al., 1992b). This longitudinal muscleportion might specifically be responsible for keepingsome tension on the scleral spur, thus preventingcollapse of Schlemm’s canal in old age (Rohen andLutjen-Drecoll, 1993). Recent studies in post-mortemhuman eyes suggests that the aging human CM is stillcapable of moving forward, albeit to a lesser degree thanin younger eyes, in response to pilocarpine (Lutjen-Drecoll, unpublished data). This could partially explainthe retention of the outflow facility response topilocarpine even in elderly subjects (Croft et al., 1996).Also changes in CM inner ring diameter in humans invivo in response to strong accommodative stimulus (8.0D) is only slightly reduced with advancing age, againsuggesting that considerable CM contractile activity andmobility persists with age (Strenk et al., 1999).An age-related decrease in Fu which occurs in rhesus
monkeys is associated with a significant increase in thethickness of the elastic fibers of the trabeculum ciliarecovering the anterior tips of the CM and an increase in
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Fig. 8. Light micrographs of sagittal sections through young (left) and old (right) rhesus monkey (top) and human (bottom) CM. Top Left: Note
that connective tissue within the CM is sparse and that the intermuscular spaces are wide, especially within the outer longitudinal portion. Top Right:
Note that the intermuscular spaces are filled with large pigmented cells. Bottom Left: The structure of the ciliary body in young human eyes resembles
that of young monkey eyes. (Azan stain). Bottom Right: An increased amount of dense connective tissue between the bundles of the intermediate
reticular CM portion is seen in elderly human but not old monkey eyes. (Crossmon stain top left and bottom right: connective tissues: green. From
Lutjen-Drecoll (1998b) with permission.
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ECM between the muscle tips (Gabelt et al., 2003). Theage-related decrease in uveoscleral drainage could alsobe due, in part to the age-related sclerosis of the CM andobliteration of the spaces between the muscle bundles(van der Zypen, 1970).In rhesus monkeys age 26–35 years, the basement
membranes of CM cells are thickened so that in someplaces, the basement membranes of adjacent cellsseemed to be fused. Intramuscular connective tissuedoes not increase until age 20 years. Spaces between theanterior tips of the outer longitudinal part of the muscleremain free of pigment up to the age of 20 years. Beyondage 20 years, the spaces between the muscle fiberbundles contain greatly increased numbers of pigmentedcells. After age 25 years, pigmented cells are present evenbetween the tips of the anterior longitudinal portion ofthe muscles (Lutjen-Drecoll et al., 1988b).In monkeys, the CM fibers show few changes with
increasing age. There is only a slight increase inextracellular material located between the musclebundles. After the age of 24 years (comparable in termsof lifespan to approximately a 70-year old human), theCM loses its ability to move anteriorly inwardly inresponse to pilocarpine, but retains the shape of arelaxed muscle (Lutjen-Drecoll et al., 1988a) (Fig. 9).The inability of the CM to move is attributed to the age-related changes in the posterior elastic tendons and their
posterior attachment zones. In young monkeys elasticfibers are homogeneous in appearance but in oldermonkeys they contain bundles of microfibrils. Theinterlacing tendons and the elastic fibers of the choroid,which contain only sparse collagen in young monkeys,become extremely densified in old monkeys (Tamm etal., 1991). An increase in collagen and fibers andpresumptive loss of elasticity of the elastic fibers, mightstiffen the posterior insertion of the CM with age(Tamm et al., 1991, 1992a). There is no age-relateddecline in CM muscarinic receptor concentration oraffinity as measured by 3H-QNB binding and noalteration in CM choline acetyltransferase or acetylcho-linesterase which might account for the inability of themuscle to respond to central or muscarinic stimuli(Gabelt et al., 1990). The decreased ability of the CM tochange shape suggests it may have less influence on theshape of the TM, which may be important formaintaining outflow through that tissue. This could, inpart, account for the decline with age in the outflowfacility response to pilocarpine in aging rhesus monkeys(Gabelt et al., 1991).
CM: glaucoma: In POAG, the amount of CM plaquematerial is significantly higher than in normal eyes.However the amount of CM plaques does not sig-nificantly correlate with age and there was no correla-tion between inner wall and CM plaques indicating that
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Fig. 9. CM topography and connective tissue distribution in rhesus monkeys. Representative sections are depicted schematically. Left, 8-year-old
rhesus monkey exhibits essentially no intramuscular connective tissue. Right, 34-year-old rhesus monkey exhibits connective tissue (arrow) only
anteriorly between longitudinal and reticular zones. Note configurational differences between atropine and pilocarpine treated muscle in the young
animal and absence of such differences in the old animals. From Lutjen-Drecoll et al., (1988a) with permission.
B.T. Gabelt, P.L. Kaufman / Progress in Retinal and Eye Research 24 (2005) 612–637 623
in glaucoma, additional factors besides age contribute toplaque formation (Lutjen-Drecoll et al., 1986a).There are no differences in the form and size of the
CM and the amount of connective tissue depo-sited between the muscle bundles in eyes withPOAG compared to age-matched controls. However,there are significant differences in the amount ofextracellular material at the anterior CM tips andtheir surrounding elastic fibers. In glaucomatouseyes, the sheath of the elastic-like fibers within thetrabeculum ciliare and the sheath surrounding theanterior elastic tendons of the CM are thickened. Theelastic-like fibers and their sheaths have a plaque-likeappearance similar to that described for the TM(Lutjen-Drecoll et al., 1986a, b). In glaucomatous eyes,the anterior tendons of the CM appear to be gluedtogether and the fiber sheaths of neighboring tendonsoften merge. Fibroblasts continuous with trabecularcells and normally surrounding the anterior muscletendons are mostly lacking in glaucomatous eyes(Lutjen-Drecoll, 1998b).In the choroid and sclera of glaucomatous eyes,
thickenings of elastic-like fiber sheaths and plate-formation are more prominent than in control eyes ofsimilar age. These structural changes at the inner surfaceof the sclera appear more pronounced than those seen inthe TM. It is not known if these changes in the fibermaterial at the border of the sclera, within the sclera and
in the emissarial scleral channels influence the perme-ability of the Fu pathways with age and glaucoma(Lutjen-Drecoll, 1998b).For a more detailed review of the morphology of the
Fu pathways, see Lutjen-Drecoll (1998b).
3.3. Ciliary epithelium
Aging: An age-dependent loss of ciliary epithelial cellsin humans has not been described (Brubaker, 1991). Inthe nonpigmented epithelium, the number of mitochon-dria increase significantly with age, and the number offenestrations in the capillary endothelium adjacent tothe ciliary epithelium increase (Hara et al., 1977; Lutjen-Drecoll, 1982). Aging nonpigmented ciliary epitheliumshows an increasing vacuolization of the cytoplasm anddeposits of lipid granules (Gartner, 1971). A decrease inmembrane infoldings (Schlotzer-Schrehardt et al., 1990;van der Zypen and Rentsch, 1971) and associated ionpumps, together with an increase of the diffusiondistance through the thickened basal stroma (Hara etal., 1977; Lutjen-Drecoll, 1982; Schlotzer-Schrehardt etal., 1990), might explain the decrease in AHF withincreasing age.
Glaucoma: In cases of end-stage glaucoma, the ciliaryprocesses show an especially extensive hyalinization ofthe stroma (Rohen and Unger, 1959). It is not known ifthese changes are due solely to aging or are the result of
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additional factors. This change could lead not only to adecrease in aqueous formation but also to a change inaqueous humor composition. This could have conse-quences for the TM establishing a vicious cycle (Lutjen-Drecoll, 1982).
4. Extracellular material or matrix components
Most hypotheses about the pathogenesis of formationof glaucomatous changes in the HTM focus on eitherthe increased synthesis of the ECM (Acott, 1992, 1994;Acott and Wirtz, 1996; Knepper et al., 1996a, b; Lutjen-Drecoll, 1998a, Lutjen-Drecoll and Rohen, 1996;Lutjen-Drecoll et al., 1986b; Murphy et al., 1992; Rohenand Witmer, 1972) or the decreased synthesis ofmetalloproteinases (Acott, 1992, 1994; Acott and Wirtz,1996; Alexander et al., 1991; Bradley et al., 1998;Samples et al., 1993). Qualitative changes in the ECM ofthe HTM could make it more resistant to pro-tease degradation, moving the balance of deposition-degradation toward accumulation. Stabilization of theECM in the HTM could have important conse-quences for aqueous humor outflow. The resiliency ofthe TM may be compromised by changes in thecomposition of the structural components of the ECM(Tripathi, 1977).
4.1. Glycosaminoglycans (GAGs) and proteoglycans
Biochemical analysis of the composition of GAGsin the TM of glaucomatous eyes reveal that hyalu-ronic acid and keratansulfate are decreased andchondroitinsulfate is increased in the glaucomatousTM (Knepper et al., 1996a, b). The total amount ofGAGs decreases in the TM of POAG eyes but the totalamount of GAG enzyme resistant material increases.One component of the enzyme resistant material thatincreases in glaucoma may be collagens like type VI(Lutjen-Drecoll, 1998a). Hyaluronic acid, which isformed by the corneal endothelium and the innertrabecular cells and washed into the TM by aqueousflow, may bind to connecting fibrils or other fibrilswithin the cribriform region (Lutjen-Drecoll et al.,1990). Hyaluronic acid covering the surfaces of theoutflow pathways might prevent adherence of moleculesto ECM components within the cribriform region andthereby prevent clogging of the outflow pathways(Lutjen-Drecoll, 1998a).An age-related, progressive coalescence of collagen
are found in normal HTM from human eye-bank eyes.The regions of collagen coalescence are associated with adecrease of small, collagen-associated proteoglycancomplexes and an increase in unrecognized matrixmaterial. (Gong et al., 1992).
4.2. Thrombospondin (TSP)
TSP-1, an extracellular matrix protein that influencescell adhesion, proliferation and angiogenesis, is also apotent endogenous activator of extracellular TGF-b.TSP-1 immunolabeling is observed throughout all layersof the TM, while other ocular tissues show no or onlyweak labeling. TSP-1 staining colocalizes with that offibronectin in the cribriform TM but not with that oftype VI collagen. In POAG eyes, TSP-1 labeling is moreintense than in normals. TSP-1 may act as a localactivator of TGF-b. In addition, incubation of TM cellswith TGF-b1 and dexamethasone increases TSP-1expression (Flugel-Koch et al., 2004).
4.3. Fibronectin
Fibronectin, an extracellular glycoprotein, is shown toplay a role in the cellular attachment to basementmembrane and cell-matrix interaction (Bornstein et al.,1978; Spiro, 1978). Quantitative morphometric evalua-tion of the trabecular drainage zone of normotensiveeyes demonstrates a slow but significant rise infibronectin content and concentration with aging.Fibronectin levels in the seventh decade and later arecomparable with those in moderately advanced andadvanced glaucoma stages. Overall, fibronectin contentis increased in advanced glaucoma (Babizhayev andBrodskaya, 1989), although a study using electronmicroscopy and immunogold labeling of basementmembranes in the TM of normal and POAG eyesshowed no increase in fibronectin in glaucoma (Hann etal., 2001). In glaucomatous eyes, fibronectin localizesmainly in the external trabecular layers, includinginterstitial spaces of the inner wall of Schlemm’s canal.If primary changes in glaucoma occur in collagenmolecules or in altered antigenic determinants of thefibronectin molecules responsible for adhesive proper-ties, decomposition of the trabecular structure couldfollow these changes (Babizhayev and Brodskaya, 1989,1993). The heparin II domain of fibronectin enhancesoutflow facility in the human anterior segment in organculture, presumably by blocking the interaction of theTM cells with the fibronectin in the ECM (Santas et al.,2003).Glaucomatous aqueous humor stimulates the migra-
tory activity of TM cells in vitro. Fibronectin accountsfor 35%–80% of the migratory activity of the aqueous.The migratory activity could explain cell loss in theaging meshwork and some of the extra loss in POAG(Hogg et al., 2000).The incidence of glaucoma is two to three times
greater in persons with diabetes than in persons ofsimilar age without diabetes (Katz and Sommer, 1988;Tielsch et al., 1995). Diabetes-associated changes in theECM of the TM may contribute to decreased aqueous
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outflow. High glucose levels in the aqueous humor mayincrease fibronectin synthesis and accumulation in theTM and accelerate the depletion of TM cells. In bovineTM cells grown in high-glucose medium, fibronectinmRNA is significantly upregulated, fibronectin immu-nofluorescence is more intense, and relative amounts offibronectin protein are significantly increased (Sato andRoy, 2002). Also the chemoattractant potential offibronectin in aqueous humor is reported to play a rolein TM cell loss in glaucoma (Hogg et al., 2000).
5. Metabolism and aqueous humor factors
Changes in gene expression resulting from elevatedIOP were studied in organ culture. Brief (6 h) elevationsin IOP in human anterior segments in organ cultureinduces selective upregulation of 11 physiologicallyrelevant genes in the TM. These include interleukin-6,preprotachykinin-1, secretogranin-II, cathespsin-L,stromelysin-1, thymosin-v4, a-tubulin, ab-crystallin,glyceraldehydes-3-phosphate dehydrogenase, metal-lothionein, and Cu/Zn superoxide dismutase. Productsof these genes are involved in vascular permeability,secretion, extracellular matrix remodeling, cytoskeletonreorganization and reactive oxygen species scavenging(Gonzalez et al., 2000). Gene expression in anteriorsegments from glaucomatous eyes in organ-culture atbaseline and in response to elevated IOP was notdetermined. It is not known if the ability to induce thesegenes is lost or perhaps long-term elevation of some ofthese gene products can be deleterious to the outflowpathways.Cross-linking of macromolecules limits their function
and fate in cells, which lead to the hypothesis that cross-linking plays a mechanistic role in the aging process(Bjorksten, 1968). Biological cross-linking reactions areenzymatically mediated or nonenzymatically activated.Nonenzymatic cross-linking activities are activated bythe Maillard glycation reaction resulting in AGEs,aldol-aldimine condensation, or by interactions withlipid peroxides. Enzymatic cross-linking activities aremediated by lysyl oxidase, tyrosine peroxidase, sulfhy-dryl oxidase, or transglutaminase (reviewed in Stitt,2001). Accumulation of cross-linked macromoleculescould impair fluid outflow pathways by mechanicalobstruction or by decreasing the compliance of theoutflow pathways, both eventually resulting in decreasedoutflow and increased IOP.
5.1. Transglutaminase
Transglutaminase enzymes catalyze posttranslationalmodification of proteins, creating covalent cross-linkswhich are resistant to disruption (Folk and Finlayson,1977). Tissue transglutaminase, the most widespread
member of this family, plays a role in cell death (Fesus etal., 1991), cell adhesion (Cai et al., 1991), andinteraction between the cell and its ECM via the cross-linking of proteins, such as fibronectin (Greenberg et al.,1991), vitronectin (Sane et al., 1988), laminin-nidogencomplexes (Aeschlimann and Paulsson, 1991; Aeschli-mann et al., 1992) and collagen type III (Bowness et al.,1987). All of these components are present in the TM(Dietlein et al., 1998; Murphy et al., 1987; Tengroth etal., 1985). Constitutive expression of tissue transgluta-minase was shown in a variety of tissues (Aeschlimannand Paulsson, 1991; Aeschlimann et al., 1992; Thomazyand Fesus, 1989). Age-related increases in transgluta-minase C activity is possibly involved in the agingprocess (Park et al., 1999). The cross-linking action oftissue transglutaminases is important not only inpathology, but also under normal conditions forpurposes of stabilizing structural proteins and ECM-cell interactions (Barsigian et al., 1991; Greenberg et al.,1991; Martinez et al., 1994). HTM was shown tosynthesize tissue transglutaminases. HTM treatmentwith TGF-b1 and b2 HTM expression of tissuetransglutaminases and fibronectin and the cross-linkingof fibronectin. This modification of the ECM in the TMhas potentially important implication for aqueoushumor dynamics in normal and glaucomatous eyessince ECM degradation by metalloproteinases could beinhibited (Welge-Lussen et al., 2000b). Since in vitrostudies reported that tissue transglutaminases enhancedconversion of latent TGF-b to active TGF-b (Kojima etal., 1993, 1995; Nunes et al., 1997), an increase in tissuetransglutaminases expression could establish a viciouscircle (Welge-Lussen et al., 2000b).
5.2. Advanced-glycation endproducts (AGE)
Advanced glycation is receiving considerable atten-tion as a possible modulator in visual disordersincluding glaucoma. An increasing number of reportsconfirm widespread advanced glycation endproduct(AGE) accumulation at sites of known ocular pathologyand demonstrate how these products mediate cross-linking of long lived molecules in the eye (Stitt, 2001).Since AGEs are constantly forming under physiologicalconditions, complex receptor systems have evolved toremove senescent, glycation modified molecules and/ordegrade existing AGE cross-links from tissues, therebylimiting their deleterious effects (Li et al., 1996; Schmidtet al., 1994; Stitt et al., 1999).Serum metabolic parameters evaluated in 49 POAG
patients without known history of diabetes mellitus and72 age and sex matched controls indicate that onlyfasting serum glucose and uric acid levels weresignificantly higher in glaucoma patients compared tocontrols. In addition, glaucoma patients have distur-bances of carbohydrate metabolism and hyperuricemia
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(Elisag et al., 2001). Changes in the chemical composi-tion, physical structure, and hydrodynamics of Bruch’smembrane were reported (Moore et al., 1995; Okubo etal., 1999; Sarks et al., 1994). AGE adducts are detectedwithin the collagenous matrix of the lamina cribrosawhere levels correlate with age (Albon et al., 2000).Inhibition of AGE formation in diabetic rats effectivelyprevents diabetes-induced myelinated optic nerve atro-phy (Ino-ue et al., 1998). Low-dose D-galactose treat-ment in mice induces AGE formation and acceleratedaging in mice (Song et al., 1999). Ultrastructural aging isproduced in the retinal pigmented epithelium-Bruch’smembrane-choriocapillaris in mice treated with low doseD-galactose, 1000� lower than those needed to inducediabetic retinopathy in mice, suggesting age-relatedchanges that promote early age-related disease (Idaet al., 2004).The eye is also susceptible to AGE formation due to
its high oxidative/photo-oxidative stress environmentand high ascorbic acid concentration (Ida et al., 2004).AGEs themselves increase oxidative stress as a result offormation of glycated proteins and binding of AGEs tospecific receptors (RAGE) generating oxidative stress,possibly through activation of transcription factorNFkB (Fu et al., 1998; Munch et al., 1996; Schmidt etal., 1996). AGEs initiate a wide range of abnormalresponses in cells and tissues, such as inappropriateexpression of growth factors, altered growth dynamics,accumulation of extracellular matrix, promotion ofvasoregulatory dysfunction, and initiation of deathpathways (Stitt et al., 1997; Stitt and Vlassara, 1999;Vlassara et al., 1994). AGEs play a role in matrixexpansion, such as in diabetic nephropathy and athero-sclerosis, by increasing expression of growth factors(TGF-b, IGF-1, PDGF-b, VEGF, CTGF), matrixproteins (collagen I, IV, laminin, fibronectin), andreducing susceptibility to matrix proteases (Mott et al.,1997; Pugliese et al., 1997, 2001; Sakaguchi et al., 2003;Twigg et al., 2002; Wendt et al., 2003; Riser et al., 2000)Riser, 2000 #3494]. CTGF is specifically stimulated byAGEs in a range of cells types in vitro (Twigg et al.,2001). Blockade or deletion of RAGE diminishes thelevels of TGFb, VEGF and other cytokines (reviewed inHudson et al., 2003) and diminishes the permeability ofvascular endothelium and inflammatory cell recruitment(Chavakis et al., 2003).The presence of these products in the outflow
pathways of the eye has not yet been establishedalthough they likely exist and contribute to theaccumulation of extracellular material and reducedelasticity as described for other parts of the eye(Farboud et al., 1999; Ida et al., 2004). Stretching isan important mechanical stimulus (Bradley et al., 2001;Mitton et al., 1997; Okada et al., 1998; Tumminia et al.,1998; WuDunn, 2001) that promotes TM remodelingpromoting the establishment of multiple preferential
flow pathways. When this stimulus is absent, ECMmaterial may accumulate in multilayer structures suchas the TM.
5.3. Hydrogen peroxide metabolism and oxidative stress
Oxidative stress contributes to the morphologic andphysiologic alterations in the aqueous outflow pathwayin aging and glaucoma (Babizhayev and Bunin, 1989;Green, 1995). The TM is in constant contact with theaqueous humor in which the key oxidant, hydrogenperoxide, is normally present (Rose et al., 1998) as aresult of reactions of ascorbic acid and trace metals(Spector and Garner, 1981). Additional hydrogenperoxide and reactive oxygen species are generated bylight-catalyzed reactions, metabolic pathways, andphagocytic or inflammatory processes (Rose et al.,1998; Spector and Garner, 1981). Hydrogen peroxideaffects aqueous outflow in the calf perfusion system(Kahn et al., 1983). HTM cells exposed to 1mMhydrogen peroxide show reduced adhesiveness to theECM proteins fibronectin, laminin, and collagen types Iand IV. The actin and vimentin structures are reorga-nized and the distribution of paxillin and focal adhesionkinase in focal contacts is reduced and the level oftranscription factor NF-kB is enhanced (Zhou et al.,1999b). Extensive and repeated oxidative stress in vivomay result in reduced TM cell adhesion, leading to cellloss which is identified as one of the major culprits inglaucomatous conditions (Alvarado et al., 1984).Oxidative DNA damage in the TM is significantly
higher in glaucoma patients than in controls (Izzotti etal., 2003). Glutathione is believed to protect oculartissues from damage induced by low hydrogen peroxideconcentrations (Costarides et al., 1991). The evidence asto whether alleles of glutathione S-transferase arepredisposing to glaucoma remains unclear (Izzotti etal., 2003, 2004; Jansson et al., 2003; Juronen et al., 2002;Wadelius, 2004).Biochemical analysis of proteins extracted from the
entire TM reveals that by the age of 80, nearly 40% ofthe protein-bound methionine is oxidized to methioninesulfoxide; a fact which might be related to the increase inlattice (curly) collagen or sheath material of the elastic-like fiber system (Horstmann et al., 1983).Aging processes in the eye occur as a consequence of
degradation of enzymes that normally metabolize anddetoxify hydrogen peroxide and other free radicals. Forexample, superoxide dismutase but not catalase specificactivity declines with age in HTM tissue extracts (De LaPaz and Epstein, 1996). Elevated levels of hydrogenperoxide and free radicals induce irreversible deleteriouseffects on different eye tissues. Glycosaminoglycansecretory patterns in the cells of the TM could bemodified (Green, 1995).
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6. Other aqueous humor factors (Table 4)
Chronic changes in the composition of factors presentin aqueous humor or vitreous could induce changesboth in the biology of the trabecular cells and in cells ofthe optic nerve head (Lutjen-Drecoll, 1998a).
6.1. Transforming growth factor b(TGFb)
TGFb2 is a component of normal aqueous humordetected in many mammalian eyes (Cousins et al., 1991;Granstein et al., 1990; Jampel et al., 1990; Tripathi et al.,1994b) and may play a dominant role in glaucomapathogenesis. The intrinsic activity of TGFb2 isconsidered to be an important factor for the main-tenance of the anterior chamber-associated immunedeviation (ACAID) (Cousins et al., 1991; Wilbanks etal., 1992).TGFb1 in aqueous humor measured by EIA is less
than 0.1 pg/ml in POAG and POAG/diabetes patientsthan in controls; total TGFb2 in diabetes+POAG issignificantly higher than control; active TGFb2 inPOAG+diabetes is significantly higher than controlsand in patients with diabetes alone (Ochiai and Ochiai,2002). Similarly increased levels of total and activeTGFb2 are found in the aqueous humor of POAGpatients compared to age-matched controls (Picht et al.,2001; Tripathi et al., 1994b). No correlation was foundbetween age and the total amount of TGFb2 in theaqueous. Also there was no correlation with the levels inthe aqueous and in the serum suggesting intraocularderivation of this cytokine (Tripathi et al., 1994a, b).TGFb2 enhancement of plasminogen activator in-
hibitor-1 expression inhibits the plasminogen/plasminsystem necessary for activation of MMPs, thus decreas-ing MMP activity and possibly contributing to increasedECM in the TM of glaucomatous eyes (Fuchshofer et
Table 4
Quantitative measures of aqueous humor factors elevated in glaucoma
al., 2003). In vitro, TGFb2 may alter ECM turnover byincreasing expression of tissue inhibitor of metallopro-teinase (Alexander et al., 1998). In human organ-cultured anterior segments, TGFb2 decreased outflowfacility and promoted focal accumulation of ECM underthe inner wall of Schlemm’s canal (Gottanka et al.,2004).TGFb2 increased irreversible cross-linking of ECM
components via action of tissue transglutminase andincreased production fibronectin in vitro (Welge-Lussenet al., 2000b).TGFbs have an inhibitory effect on the rate of cell
proliferation and motility of TM cells in vitro whichcould contribute to the decreased cellularity of the TM(Borisuth et al., 1992). TGFb isoforms significantlyinhibit EGF-stimulated TM cell proliferation in vitro(Wordinger et al., 1998). Phagocytosis is enhanced inbovine TM cells in vitro exposed to TGFb2 (Cao et al.,2003), which could also contribute to decreased TMcellularity due to rounding up and detachment of TMcells laden with phagocytosed material (Rohen and Vander Zypen, 1968; Sherwood and Richardson, 1988). Inaddition to its synthesis in various cells in the anterioreye segment, another source for TGFb that could comein contact with trabecular cells is provided by plateletsadhering to the inner wall of SC. In monkey eyes,increased IOP led to widening of intercellular clefsbetween the inner wall endothelial cells and to adherenceof platelets to these clefts (Hamanaka and Bill, 1994).HTM cells treated with TGFb expressed asm-actin. If
TGFb were released from platelets, it could possiblystimulate transformation of the TM cells to myofibro-blast-like cells (Tamm et al., 1996b). HTM cells treatedwith TGFb1 show increased expression of mRNA fortype VI-collagen subunits and increased ab-crystallinmRNA and protein (Lutjen-Drecoll, 1998a). TGFbgenerated in response to an interaction between AGEs
Specimen Ref.
Human aq Hu et al. (2002)
Human aq Tripathi et al. (1994b)
Picht et al. (2001)
Human aq Ochiai and Ochiai, (2002)
Human aq
Human TM specimens Babizhayev and Brodskaya (1989)
Human aq Tripathi et al. (1992)
Human aq Knepper et al. (2002)
ARTICLE IN PRESSB.T. Gabelt, P.L. Kaufman / Progress in Retinal and Eye Research 24 (2005) 612–637628
and RAGE could result in epithelial-myofibroblasttransdifferentiation (Oldfield et al., 2001).
6.2. ab Crystallin
In glaucomatous eyes, labeling for ab-crystallin andmyocilin occur in more regions of the TM and appearmore intense than in normal age-matched eyes (Lutjen-Drecoll et al., 1998a). In HTM cells in vitro, TGFb1 andb2 treatment increases levels of ab-crystallin expressionespecially in cells isolated from the corneoscleral TM.Cells of the cribriform region have higher basal levels ofab-crystallin. Since they are in contact with the cribri-form elastic network and therefore indirectly also withthe CM tendons, the cribriform cells may be exposed tomore mechanical stress than the corneoscleral and uvealtrabecular cells (Welge-Lussen et al., 1999). Smallincreases in ab-crystallin protein occur in CM cellsexposed to TGFb1 or TGFb2 (Welge-Lussen et al.,2000a). Heat-shock and oxidative stress increase thelevels of ab-crystallin in human and monkey TMcultures (Tamm et al., 1996a). ab-crystallin has chaper-one-like properties protecting proteins against aggrega-tion and unfolding in response to stress (Horwitz, 1992).
Expression of ELAM-1 in glaucomatous TM mayrepresent a specific marker for the disease. ELAM-1 isexpressed consistently in the TM of eyes with glaucoma,but is absent from the TM of normal eyes. Expressionresults from activation of an autocrine IL-1 feedbackloop controlled through transcription factor NF-kB,constituting a stress response similar to that found incardiovascular disease. ELAM-1 is expressed exclusivelyby glaucomatous TM but can be experimentally inducedin normal TM from human eyes by exogenous IL-1administration (Wang et al., 2001). Upregulation ofELAM-1, which is known to interact with actin(Yoshida et al., 1996) in endothelial cells, may triggerchanges in cell morphology (Kaplanski et al., 1994) thatalter cell–cell contacts, resulting in increased resistanceto aqueous outflow. IL-1 and NF-kB were initiallythought to be protective of cells subjected to stress (Begand Baltimore, 1996; Maier et al., 1990). However, thissignal would be progressively amplified by the cytokinesthrough further activation of NF-kB. Ultimately, NF-kB activation would become self-sustaining. The IL-1–NF-kB pathway generates oxygen free radicals assignaling intermediates which could cause cell damage(Uddin and Ahmad, 1995).
6.4. Vascular endothelial growth factor (VEGF)
VEGF, which stimulates the growth of vascularendothelial cells and increases vascular permeability, is
significantly increased in human eyes with glaucoma(Hu et al., 2002). No effect of age on aqueous humorVEGF concentration was detected (Hu et al., 2002).VEGF is produced by various cells types lining theanterior and posterior chambers (Babizhayev andBrodskaya, 1989) as well as other cell types in the eye.Increased levels of VEGF mRNA and protein arestimulated by hypoxia, ischemia and long-term highglucose levels (Aiello et al., 1995; Kuroki et al., 1996;Kvanta, 1995). The consequences of elevated VEGFlevels on aqueous humor dynamics have not beenestablished.
6.5. CD44S
CD44 is one type of receptor for hyaluronic acid.CD44H is an integral cell membrane glycoprotein withpostulated roles in a wide variety of biological processesincluding cell adhesion (Nondi et al., 2000), inflamma-tion (Ariel et al., 2000), autoimmunity (Gunthert andJohansson, 2000), and apoptosis (Foger et al., 2000).The soluble form CD44S is generated by the release ofthe extracellular domain by hydrolytic cleavage (Ehlersand Riordan, 1991). The concentration of solubleCD44S increases in human aqueous humor with age inboth normal aqueous and POAG aqueous but at agreater rate in POAG (Knepper et al., 2002). CD44Sshedding participates in turn-over and remodeling of theextracellular matrix and cell surface and may blockbinding of CD44H to hyaluronic acid (Ahrens et al.,2001). Knepper et al. hypothesize that POAG ischaracterized by a decreased concentration of hyaluro-nic acid and increased turnover and downregulation ofthe hyaluronic acid receptor CD44 in the eye, which, inturn, may influence cell survival of TM and retinalganglion cells (Knepper et al., 2002).
7. Future directions
The accumulation of age-related stresses throughoxidative changes and cross-linking of proteins initiatesa cascade of responses which can alter aqueous humordynamics in the eye as summarized above and in Fig. 10.Many similar responses to stress take place in POAGbut to a greater degree. An imbalance in any one orcombination of these responses either due to genetic orenvironmental factors could lead to an accumulation ofextracellular material (Gottanka et al., 1997; Lutjen-Drecoll et al., 1986a, b; Rohen and Witmer, 1972) andloss of TM cells (Alvarado et al., 1984; Grierson et al.,1982) to a point where aqueous humor outflow isimpaired (Becker, 1958; Toris et al., 2002) and IOPbecomes elevated (Armaly et al., 1980; Kini et al., 1978;Leske, 1983; Leske et al., 2003; Sommer, 1996).Therapeutic approaches to reverse the consequences
of some of these age and glaucoma induced changes that
ARTICLE IN PRESS
Fig. 10. Schematic depicting known and postulated pathways that could alter aqueous humor dynamics in aging and POAG. IGF, insulin-like
B.T. Gabelt, P.L. Kaufman / Progress in Retinal and Eye Research 24 (2005) 612–637 629
impair aqueous humor outflow are likely to have theadded benefit of reducing some of the stresses to theoutflow pathways. This is in contrast to approaches thattarget aqueous humor production. A reduction inaqueous humor production may lower IOP but theaccumulated extracellular material and levels of oxida-tive stress that the outflow pathways are subjected towould not change and could possibly increase, furtherdamaging TM and other cells and lead to furtherreductions in outflow (Becker, 1995; Kiland et al., 2004;Lutjen-Drecoll and Kaufman, 1986).Although cholinergic drug therapy has the initial
desired effect of increasing aqueous humor outflowthrough the TM (reviewed in Kaufman and Gabelt,1992), prolonged treatment can have the oppositeundesirable consequence of mechanically inducing shapechanges that may lead to underperfusion of the TM andstimulate cells to produce increased amounts of extra-cellular material (review in Lutjen-Drecoll and Kauf-man, 1993; Lutjen-Drecoll et al., 1998a, b) which candecrease outflow facility (Gabelt et al., 2004a, b).It has long been realized that outflow through the TM
can be enhanced by actin cytoskeleton altering drugs(reviewed in Kaufman, 1992; Tian et al., 2000).
However, only more recently have compounds beendiscovered that may accomplish this without toxicconsequences. The protein kinase inhibitor H-7 andthe marine macrolides latrunculin A and B increaseoutflow facility by expanding the area available for fluiddrainage in the TM (Okka et al., 2004; Peterson et al.,1999, 2000a; Tian et al., 1998). These compoundsinterfere with formation of the actin cytoskeleton orwith the cascade of events that ultimately block theinteraction of actin with myosin. In nonmuscle cells,myosin II-driven contractility is necessary for theformation and maintenance of focal adhesions andsubsequent interactions with the extracellular matrix(Geiger and Bershadsky, 2002). Expansion of the areasavailable for fluid drainage is accompanied by areduction in the amount of ECM and without apparenttoxicity to the eye in the short-term (Peterson et al.,2000b; Sabanay et al., 2000, 2004a, b; Tian et al., 2001).Other proteins along the same pathway (Fig. 11) thateventually alter actomyosin contractility have also beentargeted pharmacologically as well as with gene therapyapproaches to enhance outflow facility (Gabelt et al.,2004a; Liu et al., 2004) (Fig. 12). The physiologic andmorphologic consequences of long-term exposure to
ARTICLE IN PRESS
Fig. 11. Pathways targeting actomyosin contractility to enhance
aqueous humor outflow through the TM. MLCK, myosin light chain
Fig. 12. Fluorescence in the TM of a human anterior segment in organ
culture showing efficient transduction with an adenoviral vector
containing the fused transgene of caldesmon and green fluorescent
protein. Outflow facility was enhanced by 50% 66h after transduction.
From Gabelt et al. (2004).
B.T. Gabelt, P.L. Kaufman / Progress in Retinal and Eye Research 24 (2005) 612–637630
these agents has yet to be determined. Also, it isunknown whether age or glaucoma-related changes inthe TM cytoskeleton could alter the effectiveness ofthese compounds.Long-term expression of genes that alter aqueous
outflow has not yet been possible in the nonhumanprimate eye. Once this is accomplished, it may bepossible to determine the effects of long-term over-expression of genes that are upregulated shortly afterexposure to a stress response or elevated IOP (Borras etal., 2002). Enhancing the endogenous levels of prosta-glandin and matrix metalloproteinase synthesis mayopen up Fu pathways through the CM similar to what isaccomplished with topical prostaglandin therapies(reviewed in Weinreb et al., 2002) that are already souseful clinically to treat glaucoma (Camras et al., 1996).Identification of ways to interfere with the interaction ofTM cells with their extracellular matrix could also be a
fruitful approach for enhancing outflow (Santas et al.,2003) if components of the ECM have become excessive.Alternatively, enhancing synthesis of hyaluronic acidmay prevent accumulation of deleterious ECM compo-nents (Lutjen-Drecoll, 1998a).Further identifying and counteracting the pathways
triggered by activated TGFb could present additionaltargets for future studies. Reducing tissue transglutami-nases, PAI, asm-actin and fibronectin levels couldprevent loss of TM cells and accumulation of extra-cellular material (Alexander et al., 1998; Fuchshofer etal., 2003; Welge-Lussen et al., 2000b). The role of otheraqueous factors that are elevated in POAG such asELAM-1, CD44S, transferrin and others yet to bediscovered need to be further investigated.Finally, minimizing or reversing some of the stresses
the eye is subjected to that set in motion the detrimentalcascades of events leading to the reduction in aqueoushumor outflow could possibly be implemented aspreventive therapies after a certain age. These couldinclude enhancing the synthesis of enzymes that decreaseoxidative stress or administering compounds that breakcross-linked proteins and improving elasticity of CMattachments and TM fibers. Perhaps enhancing AHF atthe same time as outflow resistance is decreased wouldspeed the removal of stress inducing species.
Acknowledgments
EY02698, RPB, Ocular Physiology Research &Education Foundation. We thank Ernst R. Tamm,MD, for independently reviewing this work.
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