Consensus recommendations for trabecular meshwork cell isolation, characterization and culture.

Cultured trabecular meshwork (TM) cells are a valuable model system to study the cellular mechanisms involved in the regulation of conventional outflow resistance and thus intraocular pressure; and their dysfunction resulting in ocular hypertension. In this review, we describe the standard procedures used for the isolation of TM cells from several animal species including humans, and the methods used to validate their identity. Having a set of standard practices for TM cells will increase the scientific rigor when used as a model, and enable other researchers to replicate and build upon previous findings.

Induced pluripotent stem cells restore function in a human cell loss model of open-angle glaucoma.

Normally, trabecular meshwork (TM) and Schlemm's canal inner wall endothelial cells within the aqueous humor outflow pathway maintain intraocular pressure within a narrow safe range. Elevation in intraocular pressure, because of the loss of homeostatic regulation by these outflow pathway cells, is the primary risk factor for vision loss due to glaucomatous optic neuropathy. A notable feature associated with glaucoma is outflow pathway cell loss. Using controlled cell loss in ex vivo perfused human outflow pathway organ culture, we developed compelling experimental evidence that this level of cell loss compromises intraocular pressure homeostatic function. This function was restored by repopulation of the model with fresh TM cells. We then differentiated induced pluripotent stem cells (iPSCs) and used them to repopulate this cell depletion model. These differentiated cells (TM-like iPSCs) became similar to TM cells in both morphology and expression patterns. When transplanted, they were able to fully restore intraocular pressure homeostatic function. This successful transplantation of TM-like iPSCs establishes the conceptual feasibility of using autologous stem cells to restore intraocular pressure regulatory function in open-angle glaucoma patients, providing a novel alternative treatment option.

Extracellular matrix in the trabecular meshwork: intraocular pressure regulation and dysregulation in glaucoma.

The trabecular meshwork (TM) is located in the anterior segment of the eye and is responsible for regulating the outflow of aqueous humor. Increased resistance to aqueous outflow causes intraocular pressure to increase, which is the primary risk factor for glaucoma. TM cells reside on a series of fenestrated beams and sheets through which the aqueous humor flows to exit the anterior chamber via Schlemm's canal. The outer trabecular cells are phagocytic and are thought to function as a pre-filter. However, most of the outflow resistance is thought to be from the extracellular matrix (ECM) of the juxtacanalicular region, the deepest portion of the TM, and from the inner wall basement membrane of Schlemm's canal. It is becoming increasingly evident that the extracellular milieu is important in maintaining the integrity of the TM. In glaucoma, not only have ultrastructural changes been observed in the ECM of the TM, and a significant number of mutations in ECM genes been noted, but the stiffness of glaucomatous TM appears to be greater than that of normal tissue. Additionally, TGFß2 has been found to be elevated in the aqueous humor of glaucoma patients and is assumed to be involved in ECM changes deep with the juxtacanalicular region of the TM. This review summarizes the current literature on trabecular ECM as well as the development and function of the TM. Animal models and organ culture models targeting specific ECM molecules to investigate the mechanisms of glaucoma are described. Finally, the growing number of mutations that have been identified in ECM genes and genes that modulate ECM in humans with glaucoma are documented.

Segmental biomechanics of the normal and glaucomatous human aqueous outflow pathway.

The conventional aqueous outflow pathway, encompassing the trabecular meshwork (TM), juxtacanalicular connective tissue (JCT), and inner wall endothelium of Schlemm's canal (SC), governs intraocular pressure (IOP) regulation. This study targets the biomechanics of low-flow (LF) and high-flow (HF) regions within the aqueous humor outflow pathway in normal and glaucomatous human donor eyes, using a combined experimental and computational approach. LF and HF TM/JCT/SC complex tissues from normal and glaucomatous eyes underwent uniaxial tensile testing. Dynamic motion of the TM/JCT/SC complex was recorded using customized green-light optical coherence tomography during SC pressurization in cannulated anterior segment wedges. A hyperviscoelastic model quantified TM/JCT/SC complex properties. A fluid-structure interaction model simulated tissue-aqueous humor interaction. FluoSpheres were introduced into the pathway via negative pressure in the SC, with their motion tracked using two-photon excitation microscopy. Tensile test results revealed that the elastic moduli of the LF and HF regions in glaucomatous eyes are 3.5- and 1.5-fold stiffer than the normal eyes, respectively. The FE results also showed significantly larger shear moduli in the TM, JCT, and SC of the glaucomatous eyes compared to the normal subjects. The LF regions in normal eyes demonstrated larger elastic moduli compared to the HF regions in glaucomatous eyes. The resultant strain in the outflow tissues and velocity of the aqueous humor in the FSI models were in good agreement with the digital volume correlation and 3D particle image velocimetry data, respectively. This study uncovers stiffer biomechanical responses in glaucomatous eyes, with LF regions stiffer than HF regions in both normal and glaucomatous eyes. STATEMENT OF SIGNIFICANCE: This study delves into the biomechanics of the conventional aqueous outflow pathway, a crucial regulator of intraocular pressure and ocular health. By analyzing mechanical differences in low-flow and high-flow regions of normal and glaucomatous eyes, this research unveils the stiffer response in glaucomatous eyes. The distinction between regions' properties offers insights into aqueous humor outflow regulation, while the integration of experimental and computational methods enhances credibility. These findings have potential implications for disease management and present a vital step toward innovative ophthalmic interventions. This study advances our understanding of glaucoma's biomechanical basis and its broader impact on ocular health.

Comparative analysis of traction forces in normal and glaucomatous trabecular meshwork cells within a 3D, active fluid-structure interaction culture environment.

This study presents a 3D in vitro cell culture model, meticulously 3D printed to replicate the conventional aqueous outflow pathway anatomical structure, facilitating the study of trabecular meshwork (TM) cellular responses under glaucomatous conditions. Glaucoma affects TM cell functionality, leading to extracellular matrix (ECM) stiffening, enhanced cell-ECM adhesion, and obstructed aqueous humor outflow. Our model, reconstructed from polyacrylamide gel with elastic moduli of 1.5 and 21.7 kPa, is based on serial block-face scanning electron microscopy images of the outflow pathway. It allows for quantifying 3D, depth-dependent, dynamic traction forces exerted by both normal and glaucomatous TM cells within an active fluid-structure interaction (FSI) environment. In our experimental design, we designed two scenarios: a control group with TM cells observed over 20 hours without flow (static setting), focusing on intrinsic cellular contractile forces, and a second scenario incorporating active FSI to evaluate its impact on traction forces (dynamic setting). Our observations revealed that active FSI results in higher traction forces (normal: 1.83-fold and glaucoma: 2.24-fold) and shear strains (normal: 1.81-fold and glaucoma: 2.41-fold), with stiffer substrates amplifying this effect. Glaucomatous cells consistently exhibited larger forces than normal cells. Increasing gel stiffness led to enhanced stress fiber formation in TM cells, particularly in glaucomatous cells. Exposure to active FSI dramatically altered actin organization in both normal and glaucomatous TM cells, particularly affecting cortical actin stress fiber arrangement. This model while preliminary offers a new method in understanding TM cell biomechanics and ECM stiffening in glaucoma, highlighting the importance of FSI in these processes. STATEMENT OF SIGNIFICANCE: This pioneering project presents an advanced 3D in vitro model, meticulously replicating the human trabecular meshwork's anatomy for glaucoma research. It enables precise quantification of cellular forces in a dynamic fluid-structure interaction, a leap forward from existing 2D models. This advancement promises significant insights into trabecular meshwork cell biomechanics and the stiffening of the extracellular matrix in glaucoma, offering potential pathways for innovative treatments. This research is positioned at the forefront of ocular disease study, with implications that extend to broader biomedical applications.

Implementing new computational methods for the study of JCT and SC inner wall basement membrane biomechanics and hydrodynamics.

PURPOSE: The conventional aqueous outflow pathway, which includes the trabecular meshwork (TM), juxtacanalicular tissue (JCT), and the inner wall endothelium of Schlemm's canal (SC), regulates intraocular pressure (IOP) by controlling the aqueous humor outflow resistance. Despite its importance, our understanding of the biomechanics and hydrodynamics within this region remains limited. Fluid-structure interaction (FSI) offers a way to estimate the biomechanical properties of the JCT and SC under various loading and boundary conditions, providing valuable insights that are beyond the reach of current imaging techniques. METHODS: In this study, a normal human eye was fixed at a pressure of 7 mm Hg, and two radial wedges of the TM tissues, which included the SC inner wall basement membrane and JCT, were dissected, processed, and imaged using 3D serial block-face scanning electron microscopy (SBF-SEM). Four different sets of images were used to create 3D finite element (FE) models of the JCT and inner wall endothelial cells of SC with their basement membrane. The outer JCT portion was carefully removed as the outflow resistance is not in that region, leaving only the SCE inner wall and a few µm of the tissue, which does contain the resistance. An inverse iterative FE algorithm was then utilized to calculate the unloaded geometry of the JCT/SC complex at an aqueous humor pressure of 0 mm Hg. Then in the model, the intertrabecular spaces, pores, and giant vacuole contents were replaced by aqueous humor, and FSI was employed to pressurize the JCT/SC complex from 0 to 15 mm Hg. RESULTS: In the JCT/SC complex, the shear stress of the aqueous humor is not evenly distributed. Areas proximal to the inner wall of SC experience larger stresses, reaching up to 10 Pa, while those closer to the JCT undergo lower stresses, approximately 4 Pa. Within this complex, giant vacuoles with or without I-pore behave differently. Those without I-pores experience a more significant strain, around 14%, compared to those with I-pores, where the strain is roughly 9%. CONCLUSIONS: The distribution of aqueous humor wall shear stress is not uniform within the JCT/SC complex, which may contribute to our understanding of the underlying selective mechanisms in the pathway.

Dynamic traction force in trabecular meshwork cells: A 2D culture model for normal and glaucomatous states.

Glaucoma, which is associated with intraocular pressure (IOP) elevation, results in trabecular meshwork (TM) cellular dysfunction, leading to increased rigidity of the extracellular matrix (ECM), larger adhesion forces between the TM cells and ECM, and higher resistance to aqueous humor drainage. TM cells sense the mechanical forces due to IOP dynamic and apply multidimensional forces on the ECM. Recognizing the importance of cellular forces in modulating various cellular activities and development, this study is aimed to develop a 2D in vitro cell culture model to calculate the 3D, depth-dependent, dynamic traction forces, tensile/compressive/shear strain of the normal and glaucomatous human TM cells within a deformable polyacrylamide (PAM) gel substrate. Normal and glaucomatous human TM cells were isolated, cultured, and seeded on top of the PAM gel substrate with embedded FluoSpheres, spanning elastic moduli of 1.5 to 80 kPa. Sixteen-hour post-seeding live confocal microscopy in an incubator was conducted to Z-stack image the 3D displacement map of the FluoSpheres within the PAM gels. Combined with the known PAM gel stiffness, we ascertained the 3D traction forces in the gel. Our results revealed meaningfully larger traction forces in the glaucomatous TM cells compared to the normal TM cells, reaching depths greater than 10-µm in the PAM gel substrate. Stress fibers in TM cells increased with gel rigidity, but diminished when stiffness rose from 20 to 80 kPa. The developed 2D cell culture model aids in understanding how altered mechanical properties in glaucoma impact TM cell behavior and aqueous humor outflow resistance. STATEMENT OF SIGNIFICANCE: Glaucoma, a leading cause of irreversible blindness, is intricately linked to elevated intraocular pressures and their subsequent cellular effects. The trabecular meshwork plays a pivotal role in this mechanism, particularly its interaction with the extracellular matrix. This research unveils an advanced 2D in vitro cell culture model that intricately maps the complex 3D forces exerted by trabecular meshwork cells on the extracellular matrix, offering unparalleled insights into the cellular biomechanics at play in both healthy and glaucomatous eyes. By discerning the changes in these forces across varying substrate stiffness levels, we bridge the gap in understanding between cellular mechanobiology and the onset of glaucoma. The findings stand as a beacon for potential therapeutic avenues, emphasizing the gravity of cellular/extracellular matrix interactions in glaucoma's pathogenesis and setting the stage for targeted interventions in its early stages.

Biomechanics of the JCT and SC Inner Wall Endothelial Cells with Their Basement Membrane Using 3D Serial Block-Face Scanning Electron Microscopy.

BACKGROUND: More than ~70% of the aqueous humor exits the eye through the conventional aqueous outflow pathway that is comprised of the trabecular meshwork (TM), juxtacanalicular tissue (JCT), the inner wall endothelium of Schlemm's canal (SC). The flow resistance in the JCT and SC inner wall basement membrane is thought to play an important role in the regulation of the intraocular pressure (IOP) in the eye, but current imaging techniques do not provide enough information about the mechanics of these tissues or the aqueous humor in this area. METHODS: A normal human eye was perfusion-fixed and a radial wedge of the TM tissue from a high-flow region was dissected. The tissues were then sliced and imaged using serial block-face scanning electron microscopy. Slices from these images were selected and segmented to create a 3D finite element model of the JCT and SC cells with an inner wall basement membrane. The aqueous humor was used to replace the intertrabecular spaces, pores, and giant vacuoles, and fluid-structure interaction was employed to couple the motion of the tissues with the aqueous humor. RESULTS: Higher tensile stresses (0.8-kPa) and strains (25%) were observed in the basement membrane beneath giant vacuoles with open pores. The volumetric average wall shear stress was higher in SC than in JCT/SC. As the aqueous humor approached the inner wall basement membrane of SC, the velocity of the flow decreased, resulting in the formation of small eddies immediately after the flow left the inner wall. CONCLUSIONS: Improved modeling of SC and JCT can enhance our understanding of outflow resistance and funneling. Serial block-face scanning electron microscopy with fluid-structure interaction can achieve this, and the observed micro-segmental flow patterns in ex vivo perfused human eyes suggest a hypothetical mechanism.

Developing an experimental-computational workflow to study the biomechanics of the human conventional aqueous outflow pathway.

The aqueous humor actively interacts with the trabecular meshwork (TM), juxtacanalicular tissue (JCT), and Schlemm's canal (SC) through a dynamic fluid-structure interaction (FSI) coupling. Despite the fact that intraocular pressure (IOP) undergoes significant fluctuations, our understanding of the hyperviscoelastic biomechanical properties of the aqueous outflow tissues is limited. In this study, a quadrant of the anterior segment from a normal human donor eye was dynamically pressurized in the SC lumen, and imaged using a customized optical coherence tomography (OCT). The TM/JCT/SC complex finite element (FE) with embedded collagen fibrils was reconstructed based on the segmented boundary nodes in the OCT images. The hyperviscoelastic mechanical properties of the outflow tissues' extracellular matrix with embedded viscoelastic collagen fibrils were calculated using an inverse FE-optimization method. Thereafter, the 3D microstructural FE model of the TM, with adjacent JCT and SC inner wall, from the same donor eye was constructed using optical coherence microscopy and subjected to a flow load-boundary from the SC lumen. The resultant deformation/strain in the outflow tissues was calculated using the FSI method, and compared to the digital volume correlation (DVC) data. TM showed larger shear modulus (0.92 MPa) compared to the JCT (0.47 MPa) and SC inner wall (0.85 MPa). Shear modulus (viscoelastic) was larger in the SC inner wall (97.65 MPa) compared to the TM (84.38 MPa) and JCT (56.30 MPa). The conventional aqueous outflow pathway is subjected to a rate-dependent IOP load-boundary with large fluctuations. This necessitates addressing the biomechanics of the outflow tissues using hyperviscoelastic material-model. STATEMENT OF SIGNIFICANCE: While the human conventional aqueous outflow pathway is subjected to a large-deformation and time-dependent IOP load-boundary, we are not aware of any studies that have calculated the hyperviscoelastic mechanical properties of the outflow tissues with embedded viscoelastic collagen fibrils. A quadrant of the anterior segment of a normal humor donor eye was dynamically pressurized from the SC lumen with relatively large fluctuations. The TM/JCT/SC complex were OCT imaged and the mechanical properties of the tissues with embedded collagen fibrils were calculated using the inverse FE-optimization algorithm. The resultant displacement/strain in the FSI outflow model was validated versus the DVC data. The proposed experimental-computational workflow may significantly contribute to understanding of the effects of different drugs on the biomechanics of the conventional aqueous outflow pathway.

Morphological and biomechanical analyses of the human healthy and glaucomatous aqueous outflow pathway: Imaging-to-modeling.

BACKGROUND AND OBJECTIVE: Intraocular pressure (IOP) is maintained via a dynamic balance between the production of aqueous humor and its drainage through the trabecular meshwork (TM), juxtacanalicular connective tissue (JCT), and Schlemm's canal (SC) endothelium of the conventional outflow pathway. Primary open angle glaucoma (POAG) is often associated with IOP elevation that occurs due to an abnormally high outflow resistance across the outflow pathway. Outflow tissues are viscoelastic and actively interact with aqueous humor dynamics through a two-way fluid-structure interaction coupling. While glaucoma affects the morphology and stiffness of the outflow tissues, their biomechanics and hydrodynamics in glaucoma eyes remain largely unknown. This research aims to develop an image-to-model method allowing the biomechanics and hydrodynamics of the conventional aqueous outflow pathway to be studied. METHODS: We used a combination of X-ray computed tomography and scanning electron microscopy to reconstruct high-fidelity, eye-specific, 3D microstructural finite element models of the healthy and glaucoma outflow tissues in cellularized and decellularized conditions. The viscoelastic TM/JCT/SC complex finite element models with embedded viscoelastic beam elements were subjected to a physiological IOP load boundary; the stresses/strains and the flow state were calculated using fluid-structure interaction and computational fluid dynamics. RESULTS: Based on the resultant hydrodynamics parameters across the outflow pathway, the primary site of outflow resistance in healthy eyes was in the JCT and immediate vicinity of the SC inner wall, while the majority of the outflow resistance in the glaucoma eyes occurred in the TM. The TM and JCT in the glaucoma eyes showed 1.32-fold and 1.13-fold larger beam thickness and smaller trabecular space size (2.24-fold and 1.50-fold) compared to the healthy eyes. CONCLUSIONS: Characterizing the accurate morphology of the outflow tissues may significantly contribute to constructing more accurate, robust, and reliable models, that can eventually help to better understand the dynamic IOP regulation, hydrodynamics of the aqueous humor, and outflow resistance dynamic in the human eyes. This model demonstrates proof of concept for determining changes to outflow resistance in healthy and glaucomatous tissues and thus may be utilized in larger cohorts of donor tissues where disease specificity, race, age, and gender of the eye donors may be accounted for.

Modeling the Endothelial Glycocalyx Layer in the Human Conventional Aqueous Outflow Pathway.

A layer of proteoglycans and glycoproteins known as glycocalyx covers the surface of the trabecular meshwork (TM), juxtacanalicular tissue (JCT), and Schlemm's canal (SC) inner wall of the conventional aqueous outflow pathway in the eye. This has been shown to play a role in the mechanotransduction of fluid shear stress and in the regulation of the outflow resistance. The outflow resistance in the conventional outflow pathway is the main determinant of the intraocular pressure (IOP) through an active, two-way, fluid-structure interaction coupling between the outflow tissues and aqueous humor. A 3D microstructural finite element (FE) model of a healthy human eye TM/JCT/SC complex with interspersed aqueous humor was constructed. A very thin charged double layer that represents the endothelial glycocalyx layer covered the surface of the elastic outflow tissues. The aqueous humor was modeled as electroosmotic flow that is charged when it is in contact with the outflow tissues. The electrical-fluid-structure interaction (EFSI) method was used to couple the charged double layer (glycocalyx), fluid (aqueous humor), and solid (outflow tissues). When the IOP was elevated to 15 mmHg, the maximum aqueous humor velocity in the EFSI model was decreased by 2.35 mm/s (9%) compared to the fluid-structure interaction (FSI) model. The charge or electricity in the living human conventional outflow pathway generated by the charged endothelial glycocalyx layer plays a minor biomechanical role in the resultant stresses and strains as well as the hydrodynamics of the aqueous humor.

The Effect of Intraocular Pressure Load Boundary on the Biomechanics of the Human Conventional Aqueous Outflow Pathway.

BACKGROUND: Aqueous humor outflow resistance in the trabecular meshwork (TM), juxtacanalicular connective tissue (JCT), and Schlemm's canal (SC) endothelium of the conventional outflow pathway actively contribute to intraocular pressure (IOP) regulation. Outflow resistance is actively affected by the dynamic outflow pressure gradient across the TM, JCT, and SC inner wall tissues. The resistance effect implies the presence of a fluid-structure interaction (FSI) coupling between the outflow tissues and the aqueous humor. However, the biomechanical interactions between viscoelastic outflow tissues and aqueous humor dynamics are largely unknown. METHODS: A 3D microstructural finite element (FE) model of a healthy human eye TM/JCT/SC complex was constructed with elastic and viscoelastic material properties for the bulk extracellular matrix and embedded elastic cable elements. The FE models were subjected to both idealized and a physiologic IOP load boundary using the FSI method. RESULTS: The elastic material model for both the idealized and physiologic IOP load boundary at equal IOPs showed similar stresses and strains in the outflow tissues as well as pressure in the aqueous humor. However, outflow tissues with viscoelastic material properties were sensitive to the IOP load rate, resulting in different mechanical and hydrodynamic responses in the tissues and aqueous humor. CONCLUSIONS: Transient IOP fluctuations may cause a relatively large IOP difference of ~20 mmHg in a very short time frame of ~0.1 s, resulting in a rate stiffening in the outflow tissues. Rate stiffening reduces strains and causes a rate-dependent pressure gradient across the outflow tissues. Thus, the results suggest it is necessary to use a viscoelastic material model in outflow tissues that includes the important role of IOP load rate.

Viscoelastic Biomechanical Properties of the Conventional Aqueous Outflow Pathway Tissues in Healthy and Glaucoma Human Eyes.

BACKGROUND: Although the tissues comprising the ocular conventional outflow pathway have shown strong viscoelastic mechanical response to aqueous humor pressure dynamics, the viscoelastic mechanical properties of the trabecular meshwork (TM), juxtacanalicular connective tissue (JCT), and Schlemm's canal (SC) inner wall are largely unknown. METHODS: A quadrant of the anterior segment from two human donor eyes at low- and high-flow (LF and HF) outflow regions was pressurized and imaged using optical coherence tomography (OCT). A finite element (FE) model of the TM, the adjacent JCT, and the SC inner wall was constructed and viscoelastic beam elements were distributed in the extracellular matrix (ECM) of the TM and JCT to represent anisotropic collagen. An inverse FE-optimization algorithm was used to calculate the viscoelastic properties of the ECM/beam elements such that the TM/JCT/SC model and OCT imaging data best matched over time. RESULTS: The ECM of the glaucoma tissues showed significantly larger time-dependent shear moduli compared to the heathy tissues. Significantly larger shear moduli were also observed in the LF regions of both the healthy and glaucoma eyes compared to the HF regions. CONCLUSIONS: The outflow tissues in both glaucoma eyes and HF regions are stiffer and less able to respond to dynamic IOP.

Consensus Recommendation for Mouse Models of Ocular Hypertension to Study Aqueous Humor Outflow and Its Mechanisms.

Due to their similarities in anatomy, physiology, and pharmacology to humans, mice are a valuable model system to study the generation and mechanisms modulating conventional outflow resistance and thus intraocular pressure. In addition, mouse models are critical for understanding the complex nature of conventional outflow homeostasis and dysfunction that results in ocular hypertension. In this review, we describe a set of minimum acceptable standards for developing, characterizing, and utilizing mouse models of open-angle ocular hypertension. We expect that this set of standard practices will increase scientific rigor when using mouse models and will better enable researchers to replicate and build upon previous findings.

Normal and glaucomatous outflow regulation.

Glaucoma remains only partially understood, particularly at the level of intraocular pressure (IOP) regulation. Trabecular meshwork (TM) and Schlemm's canal inner wall endothelium (SCE) are key to IOP regulation and their characteristics and behavior are the focus of much investigation. This is becoming more apparent with time. We and others have studied the TM and SCE's extracellular matrix (ECM) extensively and unraveled much about its functions and role in regulating aqueous outflow. Ongoing ECM turnover is required to maintain IOP regulation and several TM ECM manipulations modulate outflow facility. We have established clearly that the outflow pathway senses sustained pressure deviations and responds by adjusting the outflow resistance correctively to keep IOP within an appropriately narrow range which will not normally damage the optic nerve. The glaucomatous outflow pathway has in many cases lost this IOP homeostatic response, apparently due at least in part, to loss of TM cells. Depletion of TM cells eliminates the IOP homeostatic response, while restoration of TM cells restores it. Aqueous outflow is not homogeneous, but rather segmental with regions of high, intermediate and low flow. In general, glaucomatous eyes have more low flow regions than normal eyes. There are distinctive molecular differences between high and low flow regions, and during the response to an IOP homeostatic pressure challenge, additional changes in segmental molecular composition occur. In conjunction with these changes, the biomechanical properties of the juxtacanalicular (JCT) segmental regions are different, with low flow regions being stiffer than high flow regions. The JCT ECM of glaucomatous eyes is around 20 times stiffer than in normal eyes. The aqueous humor outflow resistance has been studied extensively, but neither the exact molecular components that comprise the resistance nor their exact location have been established. Our hypothetical model, based on considerable available data, posits that the continuous SCE basal lamina, which lies between 125 and 500 nm beneath the SCE basal surface, is the primary source of normal resistance. On the surface of JCT cells, small and highly controlled focal degradation of its components by podosome- or invadopodia-like structures, PILS, occurs in response to pressure-induced mechanical stretching. Sub-micron sized basement membrane discontinuities develop in the SCE basement membrane and these discontinuities allow passage of aqueous humor to and through SCE giant vacuoles and pores. JCT cells then relocate versican with its highly charged glycosaminoglycan side chains into the discontinuities and by manipulation of their orientation and concentration, the JCT and perhaps the SCE cells regulate the amount of fluid passage. Testing this outflow resistance hypothesis is ongoing in our lab and has the potential to advance our understanding of IOP regulation and of glaucoma.

Gap junction connexin43 is a key element in mediating phagocytosis activity in human trabecular meshwork cells.

Human trabecular meshwork (TM) cells play pivotal roles in maintaining homeostasis of intraocular pressure via regulation of aqueous humor outflow. These cells are capable of phagocytosis, which is considered to be essential for their regulatory function. In addition, there is a strong expression of the gap junction protein connexin43 (Cx43) in the TM. Here, we investigated functional relationships between phagocytosis activity of TM cells and their expression of Cx43. Phagocytosis was measured by showing the ability of TM cells to engulf inert fluorescent particles consisting of pHrodo. We found that internalized pHrodo was partially co-localized with Cx43 and that the phagocytic activity was dramatically reduced after knockdown of Cx43 using lentiviral Cx43 shRNA. These results suggest that Cx43 is involved in the regulation of phagocytosis by TM cells.

ZO-1 associates with α3 integrin and connexin43 in trabecular meshwork and Schlemm's canal cells.

Cellular structures that perform essential homeostatic functions include tight junctions, gap junctions, desmosomes and adherens junctions. The aqueous humor, produced by the ciliary body, passes into the anterior chamber of the eye and is filtered by the trabecular meshwork (TM), a tiny tissue found in the angle of the eye. This tissue, along with Schlemm's canal (SC) inner wall cells, is thought to control intraocular pressure (IOP) homeostasis for normal, optimal vision. The actin cytoskeleton of the tissue plays a regulatory role in maintaining IOP. One of the key risk factors for primary open angle glaucoma is persistent elevation of IOP, which compromises the optic nerve. The ZO-1 (Zonula Occludens-1), extracellular matrix protein integrins, and gap junction protein connexin43 (Cx43) are widely expressed in many different cell populations. Here, we investigated the localization and interactions of ZO-1, α3 integrin, ß1 integrin, and Cx43 in cultured porcine TM and SC cells using RT-PCR, western immunoblotting and immunofluorescence labeling with confocal microscopy, along with co-immunoprecipitation. ZO-1 partially co-localized with α3 integrin, but not with ß1 integrin, and co-immunoprecipitated with Cx43, as well as with α3 integrin. The association of ZO-1 with α3 integrin and Cx43 suggests that these proteins may form a multiple protein complex in porcine TM and SC cells. Since integrins interact with the actin cytoskeleton via scaffolding proteins, these results implicate junctional and scaffolding protein ZO-1 as a potential control point in regulation of IOP to normal levels for glaucoma therapy.

Extracellular matrix turnover and outflow resistance.

Normal homeostatic adjustment of elevated intraocular pressure (IOP) involves remodeling the extracellular matrix (ECM) of the trabecular meshwork (TM). This entails sensing elevated IOP, releasing numerous activated proteinases to degrade existing ECM and concurrent biosynthesis of replacement ECM components. To increase or decrease IOP, the quantity, physical properties and/or organization of new components should be somewhat different from those replaced in order to modify outflow resistance. ECM degradation and replacement biosynthesis in the outflow pathway must be tightly controlled and focused to retain the complex structural organization of the tissue. Recently identified podosome- or invadopodia-like structures (PILS) may aid in the focal degradation of ECM and organization of replacement components.

Effects of modifiers of glycosaminoglycan biosynthesis on outflow facility in perfusion culture.

PURPOSE: Glycosaminoglycans (GAGs) have been implicated in the regulation of outflow resistance of aqueous humor flow through the trabecular meshwork (TM). Their role was further investigated by assessment of the effects of chlorate, an inhibitor of sulfation, and beta-xyloside, which provides a competitive nucleation point for addition of disaccharide units, in anterior segment perfusion culture. METHODS: Outflow facility was measured in perfused porcine and human anterior organ cultures treated with 20 or 50 mM sodium chlorate, or 1 mM beta-xyloside. Perturbation of extracellular matrix (ECM) components was assessed in paraffin-embedded sections by immunofluorescence and confocal microscopy. Parallel experiments were conducted on cultured TM cells. RESULTS: Outflow facility increased in porcine eyes with chlorate (3-fold) and beta-xyloside (3.5-fold) treatments. In human eyes, outflow increased approximately 1.5-fold and took longer (>48 hours) to occur. By confocal microscopy, immunostaining for chondroitin and heparan sulfates was observed on edges of human TM beams in nontreated eyes, with intense staining in the juxtacanalicular tissue (JCT) region. In treated eyes, staining of beam edges was severely reduced and was instead found in plaques. Chlorate treatment resulted in a striated pattern of GAG staining in the human JCT region. Fibronectin immunostaining was altered in beta-xyloside-treated eyes, whereas in cell culture, chlorate induced formation of thick fibronectin fibrils, to which tenascin C colocalized. CONCLUSIONS: Disrupting GAG chain biosynthesis increased outflow facility in perfusion culture and induced atypical ECM molecule interactions in cell culture. This study provides direct evidence of the critical role of GAG chains in regulating outflow resistance in human TM.

Extracellular matrix gene alternative splicing by trabecular meshwork cells in response to mechanical stretching.

PURPOSE: Elevated intraocular pressure (IOP), sensed as mechanical stretching by trabecular meshwork (TM) cells, triggers extracellular matrix (ECM) remodeling. In addition to changes in gene expression, alternative mRNA splicing may alter ECM protein isoforms. Changes in mRNA expression and alternative splicing of four ECM molecules in response to mechanical stretching of TM cells were investigated. METHODS: Porcine TM cells were mechanically stretched for 12, 24, or 48 hours. RNA was isolated, and RT-PCR was performed with primers that flanked alternatively spliced domains. PCR products were identified by DNA sequencing. Quantitative RT-PCR (qRT-PCR) was performed with primers positioned within nonspliced and spliced regions of the genes. RESULTS: Total levels of tenascin C, collagen type XII, and CD44 mRNA were increased, whereas versican mRNA levels were decreased in response to the mechanical stretch. In addition, each of these genes expressed alternate mRNA isoforms. Transcripts containing the fibronectin type III domain D of tenascin C, the long NC3 isoform of collagen type XII, the V1 isoform of versican, and exons v7 and v8 of CD44 all increased in response to mechanical stretching. A novel isoform of collagen type XII was observed that resulted in deletion of two exons, a frameshift, and a premature stop codon. This isoform was expressed only by stretched TM cells. CONCLUSIONS: These alternative splicing events led to the modulation of potential GAG attachment sites and other ECM-binding motifs. These changes should affect TM cell-ECM and/or protein-protein interactions during the ECM remodeling that occurs coincident with homeostatic restoration of IOP to normal.