**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 to the constant exposure to mechanical stress.

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

ces as the TM cells will lose its ability to mediate the stress response through the stress-

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

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

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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

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

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

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

dependent activation of the p38 MAPK pathway.

chronic oxidative stress.

being exposed to a sub-lethal stress [134].

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‐ ces as the TM cells will lose its ability to mediate the stress response through the stressdependent activation of the p38 MAPK pathway.
