**6. Exposure of trabecular meshwork to oxidative stress**

Another type of environmental stress that TM cells are exposed to is oxidative stress. Cells are constantly exposed to free radicals that are the by-products of normal cellular metabolism. In addition, the aqueous humor is itself a source of free radicals. Hydrogen peroxide (H2O2) is normally present in the aqueous humor and is suggested to be the key source of oxidative stress for the TM [67]. Initially, the concentration of H2O2, a reactive oxygen species (ROS), was reported to be between 25-60 µM in the aqueous humor using the dichloropheno-indopheno (DCPIP) assay [5,68-70]. However, technical issues with the DCPIP method, including the interference of ascorbic acid with the assay [70] and the spontaneous auto-oxidization of DCPIP in the presence of oxygen [71], has resulted in the re-examination of H2O2 in the aqueous humor. Different methods have indicated that H2O2 is present in the aqueous humor, but at much lower concentrations than previously thought [70,71]. An accurate concentration of H2O2 is still difficult to obtain and may vary greatly between individuals. Since cells of the TM are in direct contact with aqueous humor, these cells are exposed both intracellularly and extracellularly to oxidative stress.

Free radicals at lower concentrations are beneficial to the cell (reviewed in [72,73]). Low concentrations of ROS act as second messengers for signal transduction and gene regula‐ tion. For example, low concentrations of ROS activate the Nuclear factor kappa-B (NF-κB) transcription factor, which plays a key role in many cellular processes including inflamma‐ tion, cell proliferation, and apoptosis (reviewed in [74]) However, higher concentrations of free radicals can have negative effects on the cell (Figure 5). Free radicals can damage proteins and DNA, promote lipid peroxidation, disrupt mitochondrial function, and trigger cell death (reviewed in [73]). Cells have an antioxidant defense mechanism to counter the deleterious effects of ROS. For example, superoxide dismutase (SOD) is an antioxidant enzyme that converts superoxide free radical anion (O2 - ) into H2O2 and molecular oxygen (O2) [75]. H2O2 must then be converted into H2O by two other antioxidant enzymes: peroxisomal catalases and the family of glutathione peroxidases (GPx). In the event that H2O2 is not converted, then it may split into the hydroxyl radical (OH•), which can be dangerous because it can react with almost any macromolecule within a short diffusion distance. Cells, through the activity of nitric oxide synthase, are able to produce the free radical nitric oxide (NO ). NO itself is hardly toxic and is in fact important in regulating various cellular functions. In fact NO has been suggested to increase aqueous humor outflow by relaxing the ciliary smooth muscles [76,77]. However, NO becomes dangerous when it spontaneously reacts with superoxide O2 - , forming the powerful oxidant peroxynitrite (ONOO-) [78]. Peroxynitrite is highly reactive and can damage biological molecules resulting in cell death (reviewed in [79]). In this way, the antioxidant defense mechanism also functions in minimizing the deleterious effects of reactive nitrogen species (RNS).

Chronic oxidative stress is recognized to be a major contributor to the aging process and various diseases including neurodegenerative diseases such as Parkinson's [80,81] and Alzheimer [82-84], cancer [72,85], and cardiovascular diseases [86]. Since POAG is an agerelated disease, chronic oxidative stress is also suggested to have a role in the pathophysiology of this disease (reviewed in [87]). In POAG, both the RGCs and the anterior segment structures such as the TM are exposed to chronic oxidative stress conditions. TM cells are exposed to acute oxidative stress under normal physiological conditions [67]. The presence of cellular defense mechanisms in TM cells enables TM cells to quickly and effectively respond and adapt to their environment (Figure 2). Two cellular defense mechanisms present in TM cells are the

movements, and even with a change in body position. A supine body position has been shown to result in higher IOP compared with an upright body position [64,65]. The temporary fluctuation in IOP can vary up to 10mmHg [66]. This change in IOP results in distortions (including stretching and compression) of the cells and is sensed by the cells of the TM as

**Figure 4. Trabecular meshwork of glaucoma phenotype**. Similar to normal non-aging and aging conditions, tra‐ becular meshwork (TM) cells are exposed to a variety of stresses. However, other unknown factors are present to ini‐ tiate the cascade of events that lead to the development of glaucoma. Also, genetic mutations could compromise normal TM cell function. All of these factors are predicted to result in TM cell death (dotted circles) to the extent that the TM tissue is no longer able to function properly. Consequently, there will be dysregulation of aqueous humor drainage resulting in increased intraocular pressure (IOP), which would ultimately lead to retinal ganglion cell (RGC)

Another type of environmental stress that TM cells are exposed to is oxidative stress. Cells are constantly exposed to free radicals that are the by-products of normal cellular metabolism. In addition, the aqueous humor is itself a source of free radicals. Hydrogen peroxide (H2O2) is

**6. Exposure of trabecular meshwork to oxidative stress**

mechanical stress.

death and glaucoma.

34 Glaucoma - Basic and Clinical Aspects

antioxidant system, which defends against ROS, and the proteolytic system, which removes unwanted biomaterials from the cell, many of which are products of oxidative stress-related damage. However, as aging progresses, the normal cellular defense mechanisms become less effective and the cell is less able to remove potential toxic materials such as ROS and misfolded proteins (Figure 3). The gradual accumulation of toxic materials will lead to an environment where the cells are exposed to chronic oxidative stress. We hypothesize that the cellular defense mechanisms, already compromised due to the aging process, become completely over‐ whelmed under such chronic oxidative stress conditions. Cell death will occur when the cells are no longer able to adapt to the environment. Since accumulation of ROS occurs with age, the loss of TM cells during the aging process may also be in part due to exposure of TM cells to chronic oxidative stress conditions. The presence of fewer TM cells as aging progresses could also be detrimental to the TM tissue as there are fewer cells to protect against ROS in the aqueous humor. Although it remains a likely possibility, evidence that chronic oxidative stress directly contributes to the loss of TM cells with age is currently lacking.

droxy-2`-deoxyguanosine (8-OHdG). The levels of 8-OHdG were increased in DNA extracts from glaucomatous TM cells compared with healthy controls [88,89]. Also, aqueous humor and serum levels of 8-OHdG were significantly higher in glaucoma patients (n=28] compared

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37

Despite having more oxidative stress-related damages, TM cells of glaucoma patients appear to have increased activity of some components of the antioxidant defense mechanism. Increased levels of glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities were measured in aqueous humor of glaucomatous patients compared with the control group of senile cataract patients [94,95]. However, no apparent change in catalase activity levels have been detected [94,95]. Thus, at least some components of the antioxidant defense mechanism

Aqueous humor is both a source of free radicals and a source of antioxidants. Since low concentrations of free radicals are necessary for normal cellular function, TM cells rely on the very high content of antioxidants in the aqueous humor to achieve a balance that maximizes cell survival. High aqueous humor concentration of the antioxidant ascorbic acid (aka Vitamin C), which is about 20 times higher than in plasma [70], suggests that this antioxidant may be a major protector against free radicals in the eye [96-98]. Ascorbic acid has also been suggested to protect cells against ultraviolet light [98,99]. In addition to being an antioxidant, ascorbic acid is suggested to also have a role regulating the ECM of the TM. TM cells synthesize many types of glycosaminoglycans (GAGs) into the ECM including hyaluronic acid [100]. Ascorbic acid can increase hyaluronic acid synthesis [100]. Since hyaluronic acid has been shown to increase the expression of several MMPs [101], altered levels of this GAG would affect ECM turnover. Interestingly, Knepper *et al.* has shown that there is significantly less hyaluronic acid in the TM of POAG patients compared with the TM of normal subjects [102,103]. Thus, ascorbic acid is predicted to affect the aqueous outflow pathway by acting as an antioxidant and by regulating the ECM components that are important in maintaining the aqueous humor outflow pathway. Although some groups observed no difference in aqueous ascorbic acid levels between POAG patients and senile cataract patients [104,105], Lee *et al.* observed greater levels of ascorbic acid in the aqueous humor of POAG patients [106]. The difference in observation may be due to the great individual variation in ascorbic acid levels [105]. Nevertheless, ascorbic

In addition to the antioxidant system, the proteolytic system is another cellular defense mechanism present in TM cells. The proteolytic system is essential for the removal of oxida‐ tively damaged proteins and organelles. The 20S proteasome, 26S proteasome, and immuno‐ proteasome are the main cellular systems in eukaryotic cells that eliminate damaged proteins. The 20S proteasome tends to degrade oxidized proteins while the 26S proteasome degrade ubiquitynated proteins. In many tissues, including the TM, there is a decline in proteasomal activity with age. Caballero *et al.* reported that primary cultures of human trabecular mesh‐ work (HTM) cells from healthy older donors (ages 66, 70, and 73) had decreased proteasomal activity compared with healthy young donors (ages 9, 14, and 25) [90]. Since the overall proteasomal content did not change between the older and younger donors, the decrease in proteasomal activity is most likely due to oxidation of the proteasomal subunits and the

with the age-matched control group of senile cataract patients (n=27) [93].

are functioning to prevent TM cell death under glaucomatous conditions.

acid appears to play a protective role for TM cells.

In comparison to non-glaucomatous individuals, the TM cells of glaucoma patients appear to have more oxidative stress-related damages, including the accumulation of oxidatively damaged DNA[88,89], proteins[90], and organelles, as well as lipid peroxidation products [91,92] (Figure 5). Oxidative stress can damage DNA, resulting in the formation of 8-hy‐

**Figure 5.** Overview of various oxidative stress-related effects on the cell, which would contribute to cell death.

droxy-2`-deoxyguanosine (8-OHdG). The levels of 8-OHdG were increased in DNA extracts from glaucomatous TM cells compared with healthy controls [88,89]. Also, aqueous humor and serum levels of 8-OHdG were significantly higher in glaucoma patients (n=28] compared with the age-matched control group of senile cataract patients (n=27) [93].

antioxidant system, which defends against ROS, and the proteolytic system, which removes unwanted biomaterials from the cell, many of which are products of oxidative stress-related damage. However, as aging progresses, the normal cellular defense mechanisms become less effective and the cell is less able to remove potential toxic materials such as ROS and misfolded proteins (Figure 3). The gradual accumulation of toxic materials will lead to an environment where the cells are exposed to chronic oxidative stress. We hypothesize that the cellular defense mechanisms, already compromised due to the aging process, become completely over‐ whelmed under such chronic oxidative stress conditions. Cell death will occur when the cells are no longer able to adapt to the environment. Since accumulation of ROS occurs with age, the loss of TM cells during the aging process may also be in part due to exposure of TM cells to chronic oxidative stress conditions. The presence of fewer TM cells as aging progresses could also be detrimental to the TM tissue as there are fewer cells to protect against ROS in the aqueous humor. Although it remains a likely possibility, evidence that chronic oxidative stress

In comparison to non-glaucomatous individuals, the TM cells of glaucoma patients appear to have more oxidative stress-related damages, including the accumulation of oxidatively damaged DNA[88,89], proteins[90], and organelles, as well as lipid peroxidation products [91,92] (Figure 5). Oxidative stress can damage DNA, resulting in the formation of 8-hy‐

**Figure 5.** Overview of various oxidative stress-related effects on the cell, which would contribute to cell death.

directly contributes to the loss of TM cells with age is currently lacking.

36 Glaucoma - Basic and Clinical Aspects

Despite having more oxidative stress-related damages, TM cells of glaucoma patients appear to have increased activity of some components of the antioxidant defense mechanism. Increased levels of glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities were measured in aqueous humor of glaucomatous patients compared with the control group of senile cataract patients [94,95]. However, no apparent change in catalase activity levels have been detected [94,95]. Thus, at least some components of the antioxidant defense mechanism are functioning to prevent TM cell death under glaucomatous conditions.

Aqueous humor is both a source of free radicals and a source of antioxidants. Since low concentrations of free radicals are necessary for normal cellular function, TM cells rely on the very high content of antioxidants in the aqueous humor to achieve a balance that maximizes cell survival. High aqueous humor concentration of the antioxidant ascorbic acid (aka Vitamin C), which is about 20 times higher than in plasma [70], suggests that this antioxidant may be a major protector against free radicals in the eye [96-98]. Ascorbic acid has also been suggested to protect cells against ultraviolet light [98,99]. In addition to being an antioxidant, ascorbic acid is suggested to also have a role regulating the ECM of the TM. TM cells synthesize many types of glycosaminoglycans (GAGs) into the ECM including hyaluronic acid [100]. Ascorbic acid can increase hyaluronic acid synthesis [100]. Since hyaluronic acid has been shown to increase the expression of several MMPs [101], altered levels of this GAG would affect ECM turnover. Interestingly, Knepper *et al.* has shown that there is significantly less hyaluronic acid in the TM of POAG patients compared with the TM of normal subjects [102,103]. Thus, ascorbic acid is predicted to affect the aqueous outflow pathway by acting as an antioxidant and by regulating the ECM components that are important in maintaining the aqueous humor outflow pathway. Although some groups observed no difference in aqueous ascorbic acid levels between POAG patients and senile cataract patients [104,105], Lee *et al.* observed greater levels of ascorbic acid in the aqueous humor of POAG patients [106]. The difference in observation may be due to the great individual variation in ascorbic acid levels [105]. Nevertheless, ascorbic acid appears to play a protective role for TM cells.

In addition to the antioxidant system, the proteolytic system is another cellular defense mechanism present in TM cells. The proteolytic system is essential for the removal of oxida‐ tively damaged proteins and organelles. The 20S proteasome, 26S proteasome, and immuno‐ proteasome are the main cellular systems in eukaryotic cells that eliminate damaged proteins. The 20S proteasome tends to degrade oxidized proteins while the 26S proteasome degrade ubiquitynated proteins. In many tissues, including the TM, there is a decline in proteasomal activity with age. Caballero *et al.* reported that primary cultures of human trabecular mesh‐ work (HTM) cells from healthy older donors (ages 66, 70, and 73) had decreased proteasomal activity compared with healthy young donors (ages 9, 14, and 25) [90]. Since the overall proteasomal content did not change between the older and younger donors, the decrease in proteasomal activity is most likely due to oxidation of the proteasomal subunits and the overload of the proteasomal machinery with damaged proteins. Caballero *et al*. observed an increase in oxidized proteins in the older donors [90]. Accumulation of oxidized protein is not the only biomolecule detrimental to proteasomal function.

**8. Sensors of TM cells**

stresses such as mechanical and oxidative stresses.

to the constant exposure to mechanical stress.

Cells have stress sensors that are highly specialized for survival in a particular environment. The specific mechanism of how TM cells sense various stimuli is largely unknown [48]. Mechanosensitive ion channels, specifically calcium-dependent maxi-K+ channels, are present in TM cells [113]. Stretch-activated channels located on the TM cell membrane are predicted to increase intracellular calcium levels. Another potential mechanism through which TM cells sense mechanical stress is the ECM. ECM receptors such as integrins are connected to the cytoskeleton, which is attached to the nuclear membrane. Thus, signals may be propagated from the extracellular environment where the mechanical stress occurs to the nucleus where gene expression can be altered in response to the stress [114]. Although the consequences of oxidative stress-related damages have been extensively studied, how the cell initially senses oxidative stress remains largely unknown [48,115]. In fact, the identification of oxidative stress sensors in any cell type has proven to be very difficult. In the future, identifying more sensors in TM cells will give insight into how TM cells achieve specificity in responding to specific

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**9. Global change in gene expression in response to mechanical stress**

Exposure of TM cells to acute mechanical stress requires a quick and specific adaptive response to ensure maximal survival occurs. Recent studies examining the change in the global gene expression profile of TM cells has given insight into how TM cells are able to adapt and respond

Several groups have examined the change in global gene expression profile of TM cells in response to mechanical stress [116-119]. Vittitow *et al*. and Vittal *et al.* both observed a change in expression of a large number of genes in response to mechanical stress. In TM from postmortem human donors, application of mechanical stress resulted in the upregulation of 40 genes and the downregulation of 14 genes [116]. Mechanical stretching of cultured porcine TM cells resulted in the upregulation of 126 genes and downregulation of 29 genes [117]. However, there was very little overlap in genes between the studies most likely due to the use of different experimental models as well of stochastic factors. Nevertheless, these studies reveal that TM cells appear to respond specifically to the *type* of stress. A large number of genes that changed expression levels were involved in ECM and cytoskeletal function, which is predicted to function in response to mechanical-stretch related changed to the cell and extracellular environment [117,118]. Several studies have shown that exposure of TM cells to mechanical stress results in increased levels of active MMPs, specifically MMP2 [120-123]. The MMP family of zinc proteases initiate ECM turnover, which has been predicted to regulate aqueous humor outflow facility by altering resistance. Furthermore, temporal variation of mechanical stretching resulted in a different gene expression profile indicating that TM cells

are also able to respond specifically to the *magnitude* of mechanical stress.

Induction of stress response is thought to result in conditions that are detrimental to cell growth due in part to activation of cell cycle checkpoints [115,124]. Also, during stress response, the

The accumulation of lipid peroxidation products in the TM is suggested to also contribute to proteasomal dysfunction [107]. Lipid peroxidation occurs when a ROS attacks a polyunsatu‐ rated fatty acid, thus initiating the lipid peroxidation chain reaction, which results in highly reactive aldehydes [108,109]. Lipid peroxidation products interact with protein, which results in modification to the protein structure and activity [109]. Accumulation of lipid peroxidation end products have been observed in many neurodegenerative diseases including Alzheimer's disease [110] and Parkinson's disease [111]. In glaucoma, an increase in lipid peroxidation end products, including diene and triene conjugates, and Schiff's bases, were observed in glau‐ comatous TM tissue (n=17) and aqueous humor (n=16) compared to age-matched controls (n=13 and n=17, respectively) [91]. In addition, Fernandez-Durango *et al.* measured increased levels of the lipid peroxidation mediator, malondialdehyde (MDA), in the aqueous humor of patients with terminal cases of POAG (n=38) compared to the cataract control group (n=48) [92]. Accumulation of lipid peroxidation products is predicted to have severe consequences on the TM by modifying proteins such as calpain-1. The calpains are a family of calciumactivated non-lysosomal cysteine proteases. In glaucomatous TM tissue, aggregated and degraded calpain-1 is present, but calpain-1 activity is lower compared with normal TM tissue [107]. In the TM of glaucomatous eyes, the lipid peroxidation products isolevuglandins, specifically iso[4]levuglandin E2, modifies calpain-1, thereby inhibiting calpain-1 activity. Although the physiological function of calpain-1 in the TM remains to be elucidated, calpain-1 modified by isolevuglandins is more prone to form larger aggregates. One of the major consequences of this modification is a disruption in the proteasomal machinery. This type of malfunction of the proteasomal machinery appears to be specific to the TM and does not occur in the posterior segment of the eye. Thus, accumulation of oxidative stress-related biomole‐ cules along with a decrease in proteasomal activity with age perpetuates a vicious cycle that is postulated to greatly hinder cell survival.

### **7. Global change in gene expression in response to stress**

As reviewed in the previous sections, cells of the TM are exposed to a variety of environmental stresses. The stresses can vary in form (mechanical, phagocytic, and oxidative), magnitude, and duration (acute or chronic). The antioxidant system and the proteolytic system are effective cellular defense mechanisms that protect cells. Recent advances in technology have shown that a change in the global gene expression profile is another major part of a cell's adaptive response to stress (reviewed in [112]. The change in gene expression profile in response to stress has revealed that signal transduction pathways are a necessary means of integrating complex signals and propagating these signals to effectors. In the next section, we will examine the specific sensors and signal transduction pathways that result in an appropriate response to stress in TM cells.
