Mechanobiology of trabecular meshwork cells

Experimental Eye Research 88 (2009) 718–723

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Review

Mechanobiology of trabecular meshwork cells Darrell WuDunn* Department of Ophthalmology, Indiana University School of Medicine, 702 Rotary Circle, Indianapolis IN 46202, USA

a r t i c l e i n f o

a b s t r a c t

Article history: Received 1 July 2008 Accepted in revised form 16 November 2008 Available online 24 November 2008

Trabecular meshwork (TM) cells likely play a key role in regulating outflow facility and hence intraocular pressure. They function in a dynamic environment subjected to variations in mechanical and fluid shear forces. Because the extent of mechanical stress on the trabecular meshwork is dependent on the intraocular pressure, the behavior of TM cells under mechanical strain may suggest mechanisms for how outflow facility is regulated. Studies have demonstrated that TM cells respond in a variety of ways to mechanical loads, including increased extracellular matrix turnover, altered gene expression, cytokine release, and altered signal transduction. This review highlights some of the considerations and limitations of studying the mechanobiology of TM cells. Ó 2008 Elsevier Ltd. All rights reserved.

Keywords: trabecular meshwork biomechanics mechanical strain extracellular matrix

1. Introduction Elevated intraocular pressure is the most important risk factor for glaucoma and all current therapies for glaucoma involve the lowering of intraocular pressure. The intraocular pressure is elevated in primary open angle glaucoma because of impairment of outflow facility in the conventional outflow pathway, in which aqueous humor flows through the trabecular meshwork into Schlemm’s canal. Thus, clues to understanding the pathophysiology of primary open angle glaucoma may be found by studying the properties of the trabecular meshwork. Despite the importance of intraocular pressure in clinical practice, our knowledge of how intraocular pressure is regulated in human eyes is limited. Presumably, the outflow pathway has some homeostatic mechanism that keeps outflow facility within a certain range and thus keeps intraocular pressure under control. In perfused organ cultured whole eyes or anterior segments, increased perfusion flow rates initially cause increased pressure and decreased outflow facility. However, over several days, outflow facility increases and the pressure returns to baseline levels (Borras et al., 2002). This suggests that some compensatory mechanism exists in the trabecular outflow pathway. The basic assumption of research in this area is that cells within the trabecular meshwork must sense when the intraocular pressure is too high and then must respond to this pressure stimulus to increase outflow and thereby restore intraocular pressure to a normal level. Cells in the outflow pathway are likely to play a key role in intraocular pressure regulation. Outflow facility is altered by

* Tel.: þ1 317 278 2661; fax: þ1 317 278 1007. E-mail address: [email protected] 0014-4835/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.exer.2008.11.008

agents that alter cellular processes such as cytoskeletal organization (Gabelt et al., 2004) and matrix metalloproteinase activity (Pang et al., 2003) as well as by agents such as prostaglandin analogues and alpha-agonists that bind to cellular receptors. Thus, outflow facility is modulated by cells within the outflow pathway. 2. Biomechanics of the trabecular meshwork The trabecular meshwork undergoes constant deformations due to variations in intraocular pressure and ciliary muscle tone. The trabecular meshwork bridges the sulcus between Schwabe’s line and the scleral spur. Aqueous fluid passes through the trabecular meshwork, between the trabecular beams and perforated sheets, then through the juxtacanalicular trabecular tissue (JCT) before entering into Schlemm’s canal. Studies have shown that the trabecular meshwork is not a rigid structure but rather a dynamic 3-dimensional network that distends and compacts depending on the pressure gradient across it (Ethier, 2002; Johnstone and Grant, 1973). Most of the resistance to aqueous flow through the conventional outflow pathway occurs in the JCT and/or the adjacent inner wall of Schlemm’s canal (Johnson, 2006). Because of this resistance, when the pressure gradient across the trabecular meshwork is relatively high, the JCT and inner wall of Schlemm’s canal are pushed out towards the outer wall of Schlemm’s canal (Ethier, 2002; Johnstone and Grant, 1973). When the pressure gradient falls, the JCT and inner wall move away from the outer wall. Thus the distension of the trabecular meshwork generally correlates with the pressure gradient across it. With important exceptions noted below, intraocular pressure is proportional to the pressure gradient across the trabecular meshwork. Thus, distensions of the trabecular meshwork should generally correlate with intraocular pressure.

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Because the trabecular meshwork consists of an irregular lattice of beams, perforated sheets, and extracellular matrix substance (in the JCT), it is unclear how much mechanical stress is put on the trabecular structures as intraocular pressure rises. However the presence of elastin within the collagen beams and the elastic-like network of the JCT (Gong et al., 1989) suggest that the trabecular meshwork has elastic properties and undergoes considerable mechanical strain in response to stress (Grierson and Lee, 1975). Thus it is reasonable to assume that some degree of mechanical stretch (strain) occurs as intraocular pressure (stress) rises and that the trabecular meshwork will recoil to its normal configuration when the pressure is normalized. In addition to mechanical strain associated with rising intraocular pressure, the trabecular meshwork experiences smaller pulsatile distensions associated with the ocular pulsations (Johnstone, 2004). The choroidal circulation receives a surge of arterial blood during systole that recedes during diastole (Phillips et al., 1992). The resultant ocular pulsations can produce a cyclic variation in intraocular pressure of several millimeters of mercury. The intraocular pressure will also rise and fall intermittently during blinking or squeezing of the lid (Coleman and Trokel, 1969; Green and Luxenberg, 1979; Miller, 1967), extraocular muscle contractions associated with eye movements (Coleman and Trokel, 1969; Moses et al., 1982), manual eye rubbing, Valsalva maneuvers (Oggel et al., 1982), and other activities. These transient pressure spikes may be very large though short lived. It is unlikely that any of these frequent transient pressure elevations last long enough to elicit a significant compensatory pressure-lowering response by the trabecular meshwork, since normal pressure is rapidly restored after the stimulus is removed. However, it is possible that these transient pressure spikes contribute to general trabecular meshwork mechanical tone or health. In addition, because trabecular outflow is pressure dependent, the eye has the ability to dampen pressure spikes and reduce pressure fluctuations (Brubaker, 2003). Increased intraocular pressure due to an external pressure on the globe will result in increased flow of aqueous humor through the trabecular meshwork with a resultant compensatory reduction in intraocular pressure. It is clear that the trabecular meshwork experiences dynamic forces, both short and long term. Any hypotheses of intraocular pressure regulation must be able to differentiate transient pressure spikes from persistent pressure elevations. 3. Methods for studying the biomechanics of trabecular meshwork cells Cells of different types may be exposed to various types of mechanical forces. Any adherent cells experience some degree of mechanical strain through their attachment sites with the underlying substratum. Cells exert a small amount of contractive force on their underlying substratum and will contract substratum gels that are unfixed from rigid culture wells (Gills et al., 1998; Nakamura et al., 2002). If the substratum undergoes deformations due to tensile (stretch), compression, or torsional stress, then the overlying cells will experience these deformations through their sites of attachment. Most cell types are also exposed to fluid shear forces due to movement of fluids such as blood, lymph, interstitial fluid, or other body fluids. The cells of the trabecular meshwork most likely experience increased mechanical stress as the trabecular meshwork is distended. The amount of mechanical strain occurring in TM cells as intraocular pressure increases is difficult to determine but it is likely to be physiologically significant. However, fluid shear stress in the trabecular meshwork is thought to be low compared to inside the lumen of arterial vasculature or even within Schlemm’s canal (Ethier et al., 2004).

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Some researchers have suggested that trabecular meshwork cells may respond to changes in hydrostatic pressure (Wax et al., 2000). However, it is highly unlikely that variations in hydrostatic pressures experienced by the TM cells are of any physiological significance (Ethier and Johnson, 2006). Like almost all cell types, TM cells are essentially incompressible fluid sacs in a fluid environment. In addition, intraocular hydrostatic pressure is principally determined by atmospheric pressure, which can vary tremendously with altitude compared to the relatively small variations in intraocular pressure. Trabecular meshwork cells from several different species have been used to study the effect of mechanical strain. Some of the different results seen among various studies may reflect the differences in the animal source of the cells. In addition, age differences may be considerable among the source animals. Human eyes available to researchers are usually obtained from elderly persons whereas bovine and porcine eyes tend to come from young adults being slaughtered at an abattoir. 3.1. Mechanical strain devices: considerations and limitations Researchers have studied the effects of mechanical stress on TM cells in both perfusion organ culture and cell culture. Organ perfusion culture, first described by Douglas Johnson (Johnson and Tschumper, 1987), maintains the general architecture of the trabecular meshwork and thus may mimic the physiological conditions more closely than cell culture. However, organ culture also creates more difficulty in isolating the mechanical forces at play. For example, increasing the perfusion rate will increase the intraocular pressure in the organ and will no doubt increase the mechanical stretching of the trabecular meshwork (Borras, 2003; Bradley et al., 2001). However, the increased perfusion will also increase the fluid shear stress on the trabecular meshwork and within Schlemm’s canal, which complicates interpretation of results. In contrast, trabecular meshwork cell culture allows the researcher to select the cell type, the mechanical force and its magnitude. Assays of responses are also generally easier to perform with cell cultures. Various devices have been used to exert mechanical stress on TM cells grown in culture. With the most commonly used devices, cells are grown to confluency on an elastomer coated with extracellular matrix protein such as collagen or laminin (Liton et al., 2005a,b; WuDunn, 2001). The elastomer is stretched biaxially and cellular responses are assayed. Another type of apparatus that has been used extensively creates the mechanical stress by pushing up from below the center of a culture well or insert membrane with a small piston or bead causing the center of the membrane to bow upward (Bradley et al., 2001, 2003; Okada et al., 1998). Although these devices seem like very crude approximations of the trabecular meshwork structure, they probably are reasonable simulations at the cellular level. Some TM cells in vivo may undergo torsional strain or mechanical stretch that is not biaxial, however it is likely that the physiologic responses to various forms of mechanical stress are very similar. Most researchers have imparted a 5– 10% mechanical stretch of the substratum to create the mechanical strain of the TM cells, however, it remains unknown how much stretch actually occurs in the trabecular meshwork for a given degree of pressure elevation. It is also unclear how uniform is the mechanical stretching throughout the trabecular meshwork. Another consideration is whether to use static or cyclic mechanical stretching of the TM cells. In the eye, TM cells experience pulsatile (cyclic) variations in mechanical stress corresponding to the ocular pulsations. However, pressure variations from these pulsations are part of normal physiology and may be small compared to the potential pressure elevations seen in ocular

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hypertension or glaucoma. How much mechanical stretch corresponds to these ocular pulsations remains unknown. Static mechanical stretch is meant to mimic acute sustained elevation of intraocular pressure. However, cells subjected to static mechanical stretch may adapt to their new conditions by adjusting their attachment sites to the substratum and thereby reduce the mechanical strain. This might blunt a cellular response initially seen if the cells are stretched for extended periods. How long the cells would take to adapt to the static stretch is unclear, but trabecular meshwork cells regain their normal morphological appearance within 24 h of static stretching (Tumminia et al., 1998). In vivo, variations in mechanical stretching due to the ocular pulsations may prevent the cells from adapting easily to the increased stretch associated with elevated intraocular pressure. Vascular endothelial cells adhere better to stretched substratum if they are grown from pre-confluency under cyclic strain compared to if they are grown under static conditions (Shirinsky et al., 1989). Constant movements of the substratum may strengthen adherence of attachment points. Thus the dynamic conditions in the trabecular meshwork even at elevated intraocular pressures may help prevent readjustment of the physiologic set point for intraocular pressure. Thus it is clear that studies of mechanical stretching of TM cells are probably simplistic approximations of the dynamic environment in which TM cells normally reside. One must be cautious about interpreting the results of these studies since they may not be very representative of physiologic conditions. 4. Cellular responses to mechanical strain (Table 1) 4.1. Alterations to the extracellular matrix Aqueous humor must pass through the extracellular matrix within the JCT before reaching the inner wall of Schlemm’s canal. Acott and colleagues have shown that alterations in the composition of this extracellular matrix by matrix metalloproteinases (MMP) could affect aqueous outflow in organ perfusion culture (Bradley et al., 1998) and they have hypothesized that intraocular pressure may be regulated by modulation of extracellular matrix turnover (Acott and Kelley, 2008; Alexander et al., 1991; Bradley et al., 1998, 2001, 2003). These authors have suggested that when intraocular pressure increases, the TM cells undergo increased mechanical stretch transduced through their integrin-mediated attachments to the extracellular matrix. The TM cells respond by making the extracellular matrix more permeable to aqueous flow and thereby increase outflow facility. This response would result in lowering of intraocular pressure, reduced mechanical strain of TM cells, and return to normal extracellular matrix turnover. This feedback mechanism would thus maintain intraocular pressure in the normal range. Several studies have shown that cells increase turnover of extracellular matrix in response to mechanical strain (Bradley et al., 2001; Okada et al., 1998; WuDunn, 2001). Okada et al. (1998) found that growth phase bovine TM cells subjected to cyclic (0.45%, 30 s cycles) mechanical stretching for 72 h produced higher levels of matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-1 (TIMP-1) but no changes in MMP-9 or TIMP-2 levels. MMP-2, also known as gelatinase A, is produced at high levels by TM cells (Samples et al., 1993) and degrades type IV collagen, a main constituent of the trabecular extracellular matrix. MMP-2 activity is suppressed by TIMP-2 whereas MMP-9 activity is suppressed by TIMP-1. These results suggest that mechanical stretching induces a specific response involving MMP-2 -mediated matrix degradation. However, WuDunn (2001) found that when confluent bovine TM cells were subjected to 10% static mechanical stretch, both MMP-2 and MMP-9 activity increased over 48–72 h but neither

TIMP-1 nor TIMP-2 levels changed significantly. It may be TM cells behave differently in growth phase versus confluency. In growth phase, the cells are attached directly to the culture substratum whereas confluent cells are able to put down their own extracellular matrix. Thus their matrix turnover response may depend on the composition of the extracellular matrix already present. In addition, TM cells may respond differently to cyclic versus static mechanical stretching as described earlier. The two studies also differed in the stretching apparatus used. Acott and colleagues also looked at static mechanical stretching of porcine TM cells and found that at 24–72 h MMP-2 activity was moderately increased but MMP-3 and MMP-9 activities were unchanged (Bradley et al., 2001, 2003). In addition, TIMP-2 levels in the media were drastically reduced and cellular MT1-MMP levels were moderately increased by mechanical stretch. MT1-MMP, also known as MMP-14, is an activator of MMP-2 so the increased MT1MMP and the reduced TIMP-2 would increase MMP-2 activity. Interestingly, these changes appear to occur under translational regulation rather than an increase in transcription (Bradley et al., 2003). However, Liton et al. (2005a) found that transcription of MMP-2 was induced by 5% cyclic mechanical stretch and this increased transcription was inhibited in the presence of anti- TGFb1 antibody (Liton et al., 2005a). These studies all indicate that MMP-2 activity increases with mechanical stretching of TM cells and thus stretch-mediated extracellular matrix turnover may be a good model for intraocular pressure regulation. In addition to extracellular matrix breakdown associated with matrix metalloproteinase activity, mechanical stretching also stimulates extracellular protein production (Acott and Kelley, 2008; Borras, 2003; Chudgar et al., 2006; Keller et al., 2007; Vittal et al., 2005). Expression of connective tissue growth factor (CTGF), a secretory protein that enhances production of extracellular matrix components such as type I collagen and fibronection, was found to increase slightly after 16 h of cyclic 15% stretching of porcine TM cells (Chudgar et al., 2006). However, the mean increases in protein and transcript levels were considerably less than those seen in high pressure organ perfusion culture. The significance of this increase in CTGF is unclear. Since CTGF is also upregulated by TGF-b1, dexamethasone, and other agents and it plays a role in fibrosis, it may play a role in the development of glaucoma (Chudgar et al., 2006). The Acott group used quantitative RT-PCR to study several extracellular matrix molecules (Keller et al., 2007). They found increased mRNA levels of tenascin C, collagen type XII, and CD44 but decreased levels of versican in response to 10% mechanical stretching of porcine TM cells. In addition, they noted increased levels of alternate mRNA isoforms for these proteins as well as for the IIICS region of fibronectin gene (Vittal et al., 2005). This suggests that extracellular matrix turnover in response to increased intraocular pressure results in altered matrix components, which may affect matrix organization and cell–matrix interactions. Using microarrays to screen for changes in gene expression of porcine TM cells in response to 10% mechanical stretching for up to 48 h, Vittal et al. (2005) found increased expression of several extracellular matrix proteins, including NELL2, tenascin C, SPARC (secreted protein, acidic, cys-rich), fibronectin, laminin g1 chain, and collagen XIV (Vittal et al., 2005). The authors note that these matrix proteins have repeat domains with motifs that serve as specific binding regions and they function in structure and organization of tissue. Thus in response to mechanical stretch, they are likely involved in reorganization of the matrix and trabecular meshwork tissue. 4.2. Cytoskeleton and signal transduction Any mechanical stress on the trabecular meshwork will be sensed by the TM cells through their attachment sites to the

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Table 1 Stimulus

Conditions % (Hz)

Cell type

Response

Function

Reference

Cyclic biaxial stretch Static biaxial stretch Static biaxial stretch

0.45 (0.03) 10 10

Bovine TM Bovine TM Porcine/Human TM

[ MMP-2 and [ TIMP-1 [ MMP-2, [ MMP-9 [ MMP-2, [ MMP-14, Y TIMP-2

ECM turnover ECM turnover ECM turnover

Cyclic biaxial stretch Cyclic biaxial stretch Static biaxial stretch

5 (1) 15 (1) 10

Human TM Porcine TM Porcine TM

Static biaxial stretch

10

Porcine TM

ECM turnover ECM synthesis Altered ECM organization ECM reorganization

Static linear stretch

10

Human TM

Cyclic biaxial stretch

10 (1)

Human TM

[ MMP-2, [ CTGF [ CTGF [ tenascin C, [ collagen type XII, [ CD44, Y versican [ NELL2, [ tenascin C, [ SPARC, [ fibronection, [ laminin g1 chain, [ collagen XIV Actin cytoskeletal reorganization, Y tyrosine phosphorylation of major proteins, [ paxillin phosphorylation, Y then [ MAPK [ ATP release, [ ecto-ATPase

Okada et al., 1998 WuDunn, 2001 Bradley et al., 2003; Bradley et al., 2001 Liton et al., 2005a Chudgar et al., 2006 Keller et al., 2007

Cyclic biaxial stretch

5 (1)

Human TM

TGF-b1

Cyclic biaxial stretch

5 (1)

Human TM

Cyclic biaxial stretch Static linear stretch Increased perfusion

0.45 (0.03) 10 Constant pressure (60–70 mmHg) 0.45 (0.03)

Bovine/Porcine TM Human TM Human anterior segment Human TM

Cyclic biaxial stretch

Vittal et al., 2005

Cytoskeletal reorganization, signal transduction

Tumminia et al., 1998

Chow et al., 2007

[ IL-6

Purinergic receptormediated cell volume changes ECM turnover, ECM synthesis [ endothelial permeability

[ Prostaglandin F2a Myocilin Myocilin

[ uveoscleral outflow ? ?

Oculomedin

?

Liton et al., 2005a Gonzalez et al., 2000; Liton et al., 2005b Matsuo et al., 1996 Tamm et al., 1999 Borras et al., 2002 Fujiwara et al., 2003; Sato et al., 1999

TM, trabecular meshwork; ECM, extracellular matrix.

underlying extracellular matrix. The deformations will be transmitted to the actin cytoskeletal network that makes up the cells inner framework. It is not surprising that the actin cytoskeletal network is profoundly altered by mechanical stretching. Tumminia et al. demonstrated that stretched (10% uniaxial) human TM cells initially exhibit complex F-actin geodesic patterns compared to the more diffuse F-actin architecture of unstretched cells (Tumminia et al., 1998). By 24 h of stretching, the actin network returns to its pre-stretch configuration. These researchers also found that the cytoskeleton-associated chaperone, aB-crystallin, undergoes transient loss within 2 min of mechanical stretch and then starts recovering after 2 h of stretching (Mitton et al., 1997). Since aBcrystallin stabilizes polymerized actin (Singh et al., 2007; Wang and Spector, 1996), this rapid decline in aB-crystallin protein enables the prompt reorganization of the actin network in response to mechanical stretch. These results indicate that during mechanical stretching, the TM cell can actively reorganize its cytoskeleton to alleviate the stress. Whether cytoskeletal reorganization within TM cells has any effect on outflow facility is unclear. In addition to mediating the morphological response to mechanical stretch, the actin cytoskeleton plays a key role in signal transduction of mechanical forces (mechanotransduction) (Tumminia et al., 1998). Along with actin, tyrosine-phosphorylated proteins localize to the focal adhesion sites and may represent the first responders to mechanical stretch. Immediately after mechanical stretching begins, the levels of phosphorylation of the 6 major tyrosine-phosphorylated proteins (135, 94, 78, 52, 41, 28 kDa) decrease rapidly whereas paxillin undergoes increased phosphorylation. In addition, within 2 min of stretching, mitogen-activated protein kinase (MAPK) activity underwent a rapid transient decline and then a marked increase of 50% over baseline by 15 min of stretching. Expression of the immediate-early gene c-fos showed a similar transient decrease followed by an increase. This suggests that the MAPK signaling pathway becomes activated as cytoskeletal reorganization occurs. MAPK regulates multiple cellular activities including gene expression and apoptosis/survival, but it is still unknown what activities are relevant to the trabecular meshwork response to elevated intraocular pressure. The c-Jun N-terminal

kinase pathway did not show any variation in response to mechanical stretch. Cyclic mechanical stress induces a rapid increase in ATP release from TM cells (Chow et al., 2007). At 1 h of 10% cyclic stretching, ATP release into the media was almost 10-fold higher than controls. Ecto-ATPase activity was also significantly higher in media from stretched TM cells. Released ATP is thought to act on purinergic receptors in an autocrine/paracrine fashion, leading to alteration of cell volume. P2 purinoceptor subtype Y1 (P2Y1) agonists have been shown to increase outflow facility in perfused bovine anterior segments (Soto et al., 2005) so purinergic P2Y receptors may modulate intraocular pressure. The authors hypothesize that mechanical stretch induces ATP release, which acts on the P2Y receptors to increase outflow facility (Chow et al., 2007). The concomitant increase in ecto-ATPase activity provides a quick on/ off signal for the stimulation of P2Y receptors.

4.3. Cytokine secretion Mechanical strain has also been shown to stimulate release of several cytokines, including transforming growth factor beta-1 (TGF-b1) and interleukin-6 (IL-6) (Liton et al., 2005a,b). Among multiple roles in cellular growth and function, TGF-b1 is a potent inducer of extracellular matrix production. It is stored in its latent form in the pericellular space associated with the extracellular matrix and is released from the matrix when activated. Cyclic (5%, 1 cycle/s) mechanical stretch induces increased secretion of TGF-b1, activation of the TGF-b1 promoter, and activation of latent TGF-b1 (Liton et al., 2005a). In addition, TGF-b1 modulates the stretchinduced transcription of the MMP-2 gene and the CTGF gene (Liton et al., 2005a). Thus, TGF-b1 may initially increase outflow facility by mediating stretch-induced expression of MMP-2. However, since TGF-b1 is also a potent inducer of extracellular matrix synthesis, perhaps via CTGF, chronic stimulation of TGF-b1 may lead to matrix accumulation and impaired outflow. Interleukin-6 secretion and transcription has also been shown to be induced by cyclic mechanical stress (Gonzalez et al., 2000;

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Liton et al., 2005b). This induction is partially inhibited by antiTGF-b1 antibodies. IL-6 increases vascular permeability by reducing cell–cell associations and inducing matrix metalloproteinases. Thus, it may mediate increased outflow facility by improving flow through the inner wall of Schlemm’s canal or through the JCT. Indeed, IL-6 injected into perfused anterior segments increased outflow facility by 30% (Liton et al., 2005b). Therefore, IL-6 may be a prime mediator of intraocular pressure regulation. 4.4. Other responses Cyclic (0.45%; 30 s cycles) mechanical stretching induced production of prostaglandin F2a in bovine and porcine TM cells in growth phase, although confluent TM cells did not show significantly greater production (Matsuo et al., 1996). Prostaglandin F2a reduces intraocular pressure via uveoscleral and possible trabecular outflow pathways (Weinreb et al., 2002) so it may also have a role in maintaining intraocular pressure. Myocilin was initially discovered from cultured TM cells exposed to chronic dexamethasone (Polansky et al., 1997). Mutations in the myocilin gene have been identified in about 4% of persons with primary open angle glaucoma (Alward et al., 1998). Expression of myocilin mRNA has been shown to increase by 8– 24 h of 10% linear mechanical stretch (Tamm et al., 1999) and in high pressure organ perfusion culture after several days (Borras et al., 2002). While the function of myocilin protein is not well characterized, myocilin does appear to act similarly to matricellular proteins such as tenascin C and SPARC in that it can disrupt focal adhesions and inhibit adhesion of TM cells to the extracellular matrix if overexpressed (Shen et al., 2008). Oculomedin is a gene that was initially identified in human TM cells undergoing 0.45% cyclic mechanical stretch (Fujiwara et al., 2003; Sato et al., 1999). Oculomedin protein was only found in stretched TM cells but not unstretched cells, however, immunohistochemistry localizes the protein to the trabecular meshwork, Schlemm’s canal cells, photoreceptor cells, and corneal and conjunctival epithelium. Mutations in the oculomedin gene have been found in patients with primary open angle glaucoma (Fujiwara et al., 2003). Its function remains unknown. 5. Other biomechanical considerations Schlemm’s canal cells may also be important for intraocular pressure regulation. The inner wall of Schlemm’s canal may be the principal site of outflow resistance (Johnson, 2006) so the cells of the inner wall may control flow through the intercellular and intracellular pores of the inner wall. Some researchers are also studying the biomechanics of Schlemm’s canal cells, which are exposed to levels of shear stress that may approach arterial blood flow (Ethier, 2002; Ethier et al., 2004). 6. Conclusion An understanding of how intraocular pressure is regulated would be a tremendous step towards deciphering the pathophysiology of primary open angle glaucoma. Important clues to this understanding have been achieved by the study of TM cells under conditions that simulate the biomechanical environment of the trabecular meshwork. References Acott, T.S., Kelley, M.J., 2008. Extracellular matrix in the trabecular meshwork. Exp. Eye Res. 86, 543–561. Alexander, J.P., Samples, J.R., Van Buskirk, E.M., Acott, T.S., 1991. Expression of matrix metalloproteinases and inhibitor by human trabecular meshwork. Invest. Ophthalmol. Vis. Sci. 32, 172–180.

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