**10. Global change in gene expression in response to oxidative stress**

cell diverts energy to the adaptive response and as a result, less energy is available for cell growth-related functions [112,125]. Thus, there is a continuous balance between cell growth and stress response. In order to achieve this balance, stress responses must be transient and be temporally restricted. Consistent with this theory, exposure to mechanical stress resulted in a change in expression of a large number of stress-related genes while few growth-related genes were affected [117]. Another related characteristic of stress response is the highly reversible nature of the global change in gene expression. After removal of stress, inactivation of the stress-induced signal transduction pathway occurs, likely because constitutive activa‐ tion of stress response would be detrimental to the overall health of the cell. Although the specific reversal of the gene expression profile in TM cells after the removal of mechanical stress has not been examined, TM cells appear to physiologically return to the pre-stress state after a period of time. Perfusion of anterior segment cultures is an effective experimental model for examining TM function [126].In this model, anterior segment explants are perfused with culture medium at a constant pressure, resulting in a stable and physiologically relevant flow rate [122,127]. Using perfused human anterior segment culture, Bradley *et al.* observed that doubling the flow rate resulted in immediate doubling of IOP [122]. However, after two days, TM cells lowered outflow resistance and thus, restored the IOP to pre-stress levels even under conditions where the flow rate remained doubled. In this study, the TM cells appear to reach

Cells use multiple signal transduction pathways to integrate various input signals and coordinate an appropriate stress response. In TM cells, activation of signal transduction pathways appears to be important in mediating an appropriate stress response. Vittitow *et al.* observed that nearly 32% of genes altered in the global gene expression profile in response to mechanical stretch of TM cells functioned in various signal transduction pathways [116]. One particularly interesting signal transduction pathway is the stress-activated protein kinase (SAPK) pathways, a highly conserved signalling pathway in eukaryotes that are activated by many different environmental stresses. A rapid response to stress is essential to maximize cell survival. Thus, the SAPK pathway enables rapid phosphorylation of components of various signal transduction pathway so that a response occurs within minutes of initial exposure to the stress [128,129]. In mammals, the SAPKs are the p38 mitogenactivated protein kinases (MAPKs). There is evidence that the MAPK signal transduction pathway is involved in the TM cell response to mechanical stress [130]. In TM cells, the p38 MAPK pathway is suggested to modulate the regulation of stretch-induced cytokines such as TGF-β1 and IL-6 [131]. Thus, the p38 MAPK pathway functions in co-ordinating and regulating signal transduction pathways in response to stress. However, in primary glaucomatous TM cells, Zhang *et al.* demonstrated that the p38 MAPK pathway is unrespon‐ sive to exogenous manipulation, including the administration of Interleukin 1 (IL1), which has been shown to activate the p38 MAPK pathway in non-glaucomatous TM cells [132]. Although examination of the p38 MAPK pathway *in vivo* is required, this pathway may be unresponsive in glaucomatous TM cells because it is already maximally activated [132]. The cause of this constitutive activation remains unknown. Nevertheless, the constitutive activation of the stress-responsive MAPK pathway is predicted to have serious consequen‐

a new homeostatic condition even when the stress is not removed.

40 Glaucoma - Basic and Clinical Aspects

Despite the evidence that TM cells are exposed to oxidative stress, not much is currently known about the change in global gene expression profile in TM cells in response to chronic oxidative stress. Examining the effects of chronic oxidative stress on TM cells is especially challenging because it is difficult to experimentally create an environment where TM cells are exposed to chronic oxidative stress.

Porter *et al.* examined the global gene expression profile of phagocytically challenged TM cells under normal and acute oxidative stress conditions [133]. As avid phagocytes, TM cells are predicted to keep the aqueous humor outflow pathway clear of debris [55]. When TM cells were phagocytically challenged to *E. coli* under normal conditions, 1190 genes were upregu‐ lated and 728 genes were downregulated [133]. When TM cells were phagocytically challenged to E. coli under oxidatively stressed conditions at 40% O2, 976 genes were upregulated and 383 genes were downregulated. Although many of the altered genes were involved in immune response, cell adhesion, and regulation of ECM, there were only 6 genes that were altered in both the normal and oxidatively stressed conditions. TM cells therefore appear to have distinct gene expression profiles specific to the type of stress. Under experimental conditions, different types of stresses tend to be examined one at a time to elucidate the response of the cells to that particular stress. However, TM cells under physiological conditions are simultaneously exposed to different stresses. Studies in yeast have shown that cells are able to combine and integrate these different signals and produce an adaptive response [128]. Thus, analyzing a combination of stresses in TM cells will possibly reveal the role that cross-protection plays in these cells. Cross-protection refers to the ability of cells to become resistant to stress after first being exposed to a sub-lethal stress [134].

Transcription factors are essential regulators of signal transduction pathway components. NF-κB was identified as the transcription factor responsible for activation of many of the genes in the gene expression profile of phagocytically challenged TM cells including MMP1 and MMP3 [133]. The NF-κB transcription factor has also been shown to mediate the activation of endothelial leukocyte adhesion molecule (ELAM1) and the inflammatory cytokine IL1 [135]. ELAM1 is a cell adhesion molecule that is readily expressed in glaucom‐ atous TM cells [135-137]. Activation of ELAM1 and IL1 by the NF-κB transcription factor in response to oxidative stress promotes cell survival. However, constitutive activation of NFκB is predicted to be detrimental to cell survival and even contribute to the development of glaucoma [135]. The NF-κB transcription factor regulates the expression of numerous downstream target genes with varying functions including MMPs that regulate ECM turnover. Dysregulation of these downstream target genes is predicted to cause disrup‐ tions to TM cell function. In many situations, the altered gene expression returns to a steadystate level that is comparable to unstressed conditions even when the cells remain exposed to a particular stress [125]. As mentioned previously, activation of stress-related genes during a stress response diverts energy and resources from cell growth. Thus, situations where steady state levels are not achieved can pose a great risk to the overall health of the cell, ultimately affecting its ability to survive.

tion factor genes, *FOXC1* and *PITX2*, are associated with ARS [145-147]. How mutations in *FOXC1* and *PITX2* cause disease is not well understood. Recent findings have suggested that patients with these types of mutations may be more sensitive to environmental stresses [148,149]. The cells of the TM may be less tolerant when exposed to various stresses, resulting in dysregulation of aqueous humor outflow. In this section, we will take a closer look at the effects of environmental stresses on two genes, *MYOC*, which is associated with POAG, and

Genetics and Environmental Stress Factor Contributions to Anterior Segment Malformations and Glaucoma

http://dx.doi.org/10.5772/54653

43

*MYOC* was the first POAG gene to be reported [150-152]. Patients with *MYOC* mutations tend to present with juvenile and early adult-onset forms of POAG. However, the most commonly reported MYOC mutation, Q368X, is associated with later adult-onset POAG. MYOC is expressed in most ocular tissues [153] including the TM and is secreted into the aqueous humor [154,155]. The release of MYOC is associated with the release of exosomes. Signaling molecules within these exosomes is predicted to function in maintaining TM homeostasis [156]. Specific MYOC mutations appear to sensitize cells to oxidative stress. Joe *et al*. observed that Human Embryonic Kidney 293 (HEK293) cells stably transfected with the Y437H MYOC mutation have decreased expression of antioxidant genes and produced more ROS [156,157]. Also, more H2O2-induced cell death occurred in HEK293 cells overexpressing various *MYOC* mutations compared with wild type. The extent of cell death differed between mutants. Furthermore, 18 month old Y437H mutant mice had increased expression of ER stress markers and decreased levels of antioxidant proteins [157]. These findings suggest that patients with *MYOC* mutations are more sensitive to oxidative stress. The decreased ability to response to oxidative stress may

contribute to the development of glaucoma earlier on in these patients' lives.

Anterior segment dysgenesis covers a wide spectrum of developmental anomalies that can affect the iris, cornea, lens, TM, and Schlemm's canal. We have already discussed the impor‐ tance of the TM and Schlemm's canal in maintaining the aqueous humor outflow pathway. Disruptions in this pathway may result in increased IOP, which is a major risk factor of developing glaucoma. Glaucoma is estimated to develop in approximately 50% of patients with anterior segment dysgenesis [158-160]. Although the mechanism that leads to glaucoma may vary between different anterior segment dysgenesis disorders and even between indi‐ viduals with the same disorder, recent findings suggest that environmental stresses affect the normal functioning of the disease-associated gene. Patients with ARS can present with a variety of ocular anomalies and systemic anomalies. Ocular anomalies include iris hypoplasia, corectopia, polycoria, and posterior embryotoxon while systemic anomalies include dental anomalies and redundant periumbilical skin. ARS patients with FOXC1 mutations have a 50-80% risk of developing earlier-onset glaucoma [161]. As a transcription factor, FOXC1 regulates the expression of a myriad of genes including genes that function in proteolysis, cell matrix adhesion, apoptosis, signal transduction, and stress response [148]. Berry *et al.* observed that FOXC1 plays a role in TM cell viability by directly regulating the transcription factor FOXO1A which is involved in the cellular stress response pathway and apoptosis. Decreasing the expression of FOXC1 increased the sensitivity of TM cells to oxidative stress. Tight regulation of the FOXC1 transcription factor is essential because both a high (FOXC1 dupli‐ cations) and low FOXC1 (loss of function mutations) gene dose results in anterior segment

*FOXC1*, which is associated with ARS.

### **11. Unspecific gene expression response**

Studyingthe change intheglobalgene expressionprofileofTMcells reveals that a largenumber ofgenesareeitherupregulatedordownregulatedinresponsetovariousenvironmental stresses. Many of the genes that have altered expression do not appear to have any relevant function in the adaptive response to the specific stress. Analysis of global gene expression profiles in other systems such as *S. cerevisiae* yield similar findings of an unspecific gene expression response [138-140].Furthermore,studiesin*E.coli*haverevealedthatwhencellsareexposedtoanunknown stress that the cell would not encounter under normal biological conditions, an unspecific and stochastic gene expression response was triggered [141,142]. Since cells may not have specific sensors to detect multiple stresses simultaneously, the unspecific stress response has been suggestedtoprotect cellsunder multiple stress conditions by changing the expression of a large number of genes, some of which function in promoting a general adaptive response [125]. Furthermore, this unspecific and stochastic gene expression response may be an important evolutionary mechanism, thereby allowing cells to adapt to an unpredictable challenge [125]. Even though cells of the TM are in a dynamic environment with a multitude of challenges, the unspecific gene expression response may enable these cells to quickly and effectively adapt to a new steady state. In the future, distinguishing between stress-essential genes (necessary for immediate response) and stress-induced genes (most likely necessary for unspecific or longterm response) in TM cells is critical in understanding how TM cells adapt to stress in the longterm and prepare for subsequent stresses. Finally, although examining a particular stressinduced gene is important in elucidating its role in aqueous humor regulation, examining the *network*ofgenes alteredinresponse tostresswillprovide furtherinsightintothe complexnature of the adaptive response of TM cells.

### **12. Effect of environmental stress on glaucoma-associated genes**

Exposure of anterior segment structures, specifically the TM, to environmental stresses disrupts the aqueous humor outflow pathway and contributes to the development of glauco‐ ma. Glaucoma, however, has a complex etiology. In addition to the environmental stress factors, genetic factors contribute to the development of this disease. At least 14 chromosomal loci have been identified for POAG (GLC1A to GLC1N) [143]. Currently three genes from these loci have been associated with glaucoma: *myocilin* (*MYOC*), *optineurin* (*OPTN*), and *WD repeat domain 36* (*WDR36*). Mutations in these three genes account for less than 5% of POAG cases [144]. Glaucoma is also a major consequence for many anterior segment dysgenesis disorders including Axenfeld-Rieger Syndrome (ARS) and Peter's anomaly. Mutations in the transcrip‐ tion factor genes, *FOXC1* and *PITX2*, are associated with ARS [145-147]. How mutations in *FOXC1* and *PITX2* cause disease is not well understood. Recent findings have suggested that patients with these types of mutations may be more sensitive to environmental stresses [148,149]. The cells of the TM may be less tolerant when exposed to various stresses, resulting in dysregulation of aqueous humor outflow. In this section, we will take a closer look at the effects of environmental stresses on two genes, *MYOC*, which is associated with POAG, and *FOXC1*, which is associated with ARS.

to a particular stress [125]. As mentioned previously, activation of stress-related genes during a stress response diverts energy and resources from cell growth. Thus, situations where steady state levels are not achieved can pose a great risk to the overall health of the cell,

Studyingthe change intheglobalgene expressionprofileofTMcells reveals that a largenumber ofgenesareeitherupregulatedordownregulatedinresponsetovariousenvironmental stresses. Many of the genes that have altered expression do not appear to have any relevant function in the adaptive response to the specific stress. Analysis of global gene expression profiles in other systems such as *S. cerevisiae* yield similar findings of an unspecific gene expression response [138-140].Furthermore,studiesin*E.coli*haverevealedthatwhencellsareexposedtoanunknown stress that the cell would not encounter under normal biological conditions, an unspecific and stochastic gene expression response was triggered [141,142]. Since cells may not have specific sensors to detect multiple stresses simultaneously, the unspecific stress response has been suggestedtoprotect cellsunder multiple stress conditions by changing the expression of a large number of genes, some of which function in promoting a general adaptive response [125]. Furthermore, this unspecific and stochastic gene expression response may be an important evolutionary mechanism, thereby allowing cells to adapt to an unpredictable challenge [125]. Even though cells of the TM are in a dynamic environment with a multitude of challenges, the unspecific gene expression response may enable these cells to quickly and effectively adapt to a new steady state. In the future, distinguishing between stress-essential genes (necessary for immediate response) and stress-induced genes (most likely necessary for unspecific or longterm response) in TM cells is critical in understanding how TM cells adapt to stress in the longterm and prepare for subsequent stresses. Finally, although examining a particular stressinduced gene is important in elucidating its role in aqueous humor regulation, examining the *network*ofgenes alteredinresponse tostresswillprovide furtherinsightintothe complexnature

**12. Effect of environmental stress on glaucoma-associated genes**

Exposure of anterior segment structures, specifically the TM, to environmental stresses disrupts the aqueous humor outflow pathway and contributes to the development of glauco‐ ma. Glaucoma, however, has a complex etiology. In addition to the environmental stress factors, genetic factors contribute to the development of this disease. At least 14 chromosomal loci have been identified for POAG (GLC1A to GLC1N) [143]. Currently three genes from these loci have been associated with glaucoma: *myocilin* (*MYOC*), *optineurin* (*OPTN*), and *WD repeat domain 36* (*WDR36*). Mutations in these three genes account for less than 5% of POAG cases [144]. Glaucoma is also a major consequence for many anterior segment dysgenesis disorders including Axenfeld-Rieger Syndrome (ARS) and Peter's anomaly. Mutations in the transcrip‐

ultimately affecting its ability to survive.

42 Glaucoma - Basic and Clinical Aspects

of the adaptive response of TM cells.

**11. Unspecific gene expression response**

*MYOC* was the first POAG gene to be reported [150-152]. Patients with *MYOC* mutations tend to present with juvenile and early adult-onset forms of POAG. However, the most commonly reported MYOC mutation, Q368X, is associated with later adult-onset POAG. MYOC is expressed in most ocular tissues [153] including the TM and is secreted into the aqueous humor [154,155]. The release of MYOC is associated with the release of exosomes. Signaling molecules within these exosomes is predicted to function in maintaining TM homeostasis [156]. Specific MYOC mutations appear to sensitize cells to oxidative stress. Joe *et al*. observed that Human Embryonic Kidney 293 (HEK293) cells stably transfected with the Y437H MYOC mutation have decreased expression of antioxidant genes and produced more ROS [156,157]. Also, more H2O2-induced cell death occurred in HEK293 cells overexpressing various *MYOC* mutations compared with wild type. The extent of cell death differed between mutants. Furthermore, 18 month old Y437H mutant mice had increased expression of ER stress markers and decreased levels of antioxidant proteins [157]. These findings suggest that patients with *MYOC* mutations are more sensitive to oxidative stress. The decreased ability to response to oxidative stress may contribute to the development of glaucoma earlier on in these patients' lives.

Anterior segment dysgenesis covers a wide spectrum of developmental anomalies that can affect the iris, cornea, lens, TM, and Schlemm's canal. We have already discussed the impor‐ tance of the TM and Schlemm's canal in maintaining the aqueous humor outflow pathway. Disruptions in this pathway may result in increased IOP, which is a major risk factor of developing glaucoma. Glaucoma is estimated to develop in approximately 50% of patients with anterior segment dysgenesis [158-160]. Although the mechanism that leads to glaucoma may vary between different anterior segment dysgenesis disorders and even between indi‐ viduals with the same disorder, recent findings suggest that environmental stresses affect the normal functioning of the disease-associated gene. Patients with ARS can present with a variety of ocular anomalies and systemic anomalies. Ocular anomalies include iris hypoplasia, corectopia, polycoria, and posterior embryotoxon while systemic anomalies include dental anomalies and redundant periumbilical skin. ARS patients with FOXC1 mutations have a 50-80% risk of developing earlier-onset glaucoma [161]. As a transcription factor, FOXC1 regulates the expression of a myriad of genes including genes that function in proteolysis, cell matrix adhesion, apoptosis, signal transduction, and stress response [148]. Berry *et al.* observed that FOXC1 plays a role in TM cell viability by directly regulating the transcription factor FOXO1A which is involved in the cellular stress response pathway and apoptosis. Decreasing the expression of FOXC1 increased the sensitivity of TM cells to oxidative stress. Tight regulation of the FOXC1 transcription factor is essential because both a high (FOXC1 dupli‐ cations) and low FOXC1 (loss of function mutations) gene dose results in anterior segment dysgenesis phenotypes associated with glaucoma. Interestingly, FOXC1 itself appears to be responsive to stress as well (Y.A.I. and M.A.W. personal observations). Thus, the FOXC1 transcription factor appears to play an important role in responding to environmental stresses. Disruptions to normal FOXC1 function are predicted to disrupt the regulation of downstream target genes that are involved in executing a rapid and effective adaptive response. Therefore, ARS patients with FOXC1 mutations may have a compromised ability to respond to environ‐ mental stresses resulting in the early age of development of glaucoma. Thus, even in the case of anterior segment developmental disorders, oxidative stress appears to have an impact on the TM. Studying genes that are associated with both the primary and secondary glaucomas provide an invaluable tool to understanding the contribution of environmental stresses on the development of glaucoma.

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