Impaired TRPV4-eNOS signaling in trabecular meshwork elevates intraocular pressure in glaucoma.

Primary Open Angle Glaucoma (POAG) is the most common form of glaucoma that leads to irreversible vision loss. Dysfunction of trabecular meshwork (TM) tissue, a major regulator of aqueous humor (AH) outflow resistance, is associated with intraocular pressure (IOP) elevation in POAG. However, the underlying pathological mechanisms of TM dysfunction in POAG remain elusive. In this regard, transient receptor potential vanilloid 4 (TRPV4) cation channels are known to be important Ca2+ entry pathways in multiple cell types. Here, we provide direct evidence supporting Ca2+ entry through TRPV4 channels in human TM cells and show that TRPV4 channels in TM cells can be activated by increased fluid flow/shear stress. TM-specific TRPV4 channel knockout in mice elevated IOP, supporting a crucial role for TRPV4 channels in IOP regulation. Pharmacological activation of TRPV4 channels in mouse eyes also improved AH outflow facility and lowered IOP. Importantly, TRPV4 channels activated endothelial nitric oxide synthase (eNOS) in TM cells, and loss of eNOS abrogated TRPV4-induced lowering of IOP. Remarkably, TRPV4-eNOS signaling was significantly more pronounced in TM cells compared to Schlemm's canal cells. Furthermore, glaucomatous human TM cells show impaired activity of TRPV4 channels and disrupted TRPV4-eNOS signaling. Flow/shear stress activation of TRPV4 channels and subsequent NO release were also impaired in glaucomatous primary human TM cells. Together, our studies demonstrate a central role for TRPV4-eNOS signaling in IOP regulation. Our results also provide evidence that impaired TRPV4 channel activity in TM cells contributes to TM dysfunction and elevated IOP in glaucoma.

An ex vivo model of human corneal rim perfusion organ culture.

The human anterior segment perfusion culture model is a valuable tool for studying the trabecular meshwork (TM) and aqueous humor outflow in glaucoma. The traditional model relies on whole eye globes resulting in high cost and limited availability. Here, we developed a glue-based method which enabled us to use human corneal rims for perfusion culture experiments. Human corneal rim perfusion culture plates were 3D printed. Human corneal rims containing intact TM were attached and sealed to the plate using low viscosity and high viscosity glues, respectively. The human corneal rims were perfused using the constant flow mode, and the pressure changes were recorded using a computerized system. Outflow facility, TM stiffness, and TM morphology were evaluated. When perfused at rates from 1.2 to 3.6 µl/min, the outflow facility was 0.359 ± 0.216 µl/min/mmHg among 10 human corneal rims. The stiffness of the TM in naïve human corneal rim was similar to that of perfusion cultured human corneal rim. Also, the stiffness of TM of corneal rims perfused with dexamethasone was significantly higher than the control. Human corneal rims with glue contamination in the TM could be differentiated by high baseline intraocular pressure as well as high TM stiffness. Histology studies showed that the TM tissues perfused with plain medium appeared normal. We believed that our glued-based method is a useful tool and low-cost alternative to the traditional anterior segment perfusion culture model.

Technological advances in ocular trabecular meshwork in vitro models for glaucoma research.

Glaucoma is the leading cause of irreversible blindness worldwide and is characterized by the progressive degeneration of the optic nerve. Intraocular pressure (IOP), which is considered to be the main risk factor for glaucoma development, builds up in response to the resistance (resistance to what?) provided by the trabecular meshwork (TM) to aqueous humor (AH) outflow. Although the TM and its relationship to AH outflow have remained at the forefront of scientific interest, researchers remain uncertain regarding which mechanisms drive the deterioration of the TM. Current tissue-engineering fabrication techniques have come up with promising approaches to successfully recreate the TM. Nonetheless, more accurate models are needed to understand the factors that make glaucoma arise. In this review, we provide a chronological evaluation of the technological milestones that have taken place in the field of glaucoma research, and we conduct a comprehensive comparison of available TM fabrication technologies. Additionally, we also discuss AH perfusion platforms, since they are essential for the validation of these scaffolds, as well as pressure-outflow relationship studies and the discovery of new IOP-reduction therapies.

Adipose-derived stem cells integrate into trabecular meshwork with glaucoma treatment potential.

The trabecular meshwork (TM) is an ocular tissue that maintains intraocular pressure (IOP) within a physiologic range. Glaucoma patients have reduced TM cellularity and, frequently, elevated IOP. To establish a stem cell-based approach to restoring TM function and normalizing IOP, human adipose-derived stem cells (ADSCs) were induced to differentiate to TM cells in vitro. These ADSC-TM cells displayed a TM cell-like genotypic profile, became phagocytic, and responded to dexamethasone stimulation, characteristic of TM cells. After transplantation into naive mouse eyes, ADSCs and ADSC-TM cells integrated into the TM tissue, expressed TM cell markers, and maintained normal IOP, outflow facility, and extracellular matrix. Cell migration and affinity results indicated that the chemokine pair CXCR4/SDF1 may play an important role in ADSC-TM cell homing. Our study demonstrates the possibility of applying autologous or allogeneic ADSCs and ADSC-TM cells as a potential treatment to restore TM structure and function in glaucoma.

Identification of Estrogen Signaling in a Prioritization Study of Intraocular Pressure-Associated Genes.

Elevated intraocular pressure (IOP) is the only modifiable risk factor for primary open-angle glaucoma (POAG). Herein we sought to prioritize a set of previously identified IOP-associated genes using novel and previously published datasets. We identified several genes for future study, including several involved in cytoskeletal/extracellular matrix reorganization, cell adhesion, angiogenesis, and TGF-ß signaling. Our differential correlation analysis of IOP-associated genes identified 295 pairs of 201 genes with differential correlation. Pathway analysis identified ß-estradiol as the top upstream regulator of these genes with ESR1 mediating 25 interactions. Several genes (i.e., EFEMP1, FOXC1, and SPTBN1) regulated by ß-estradiol/ESR1 were highly expressed in non-glaucomatous human trabecular meshwork (TM) or Schlemm's canal (SC) cells and specifically expressed in TM/SC cell clusters defined by single-cell RNA-sequencing. We confirmed ESR1 gene and protein expression in human TM cells and TM/SC tissue with quantitative real-time PCR and immunofluorescence, respectively. 17ß-estradiol was identified in bovine, porcine, and human aqueous humor (AH) using ELISA. In conclusion, we have identified estrogen receptor signaling as a key modulator of several IOP-associated genes. The expression of ESR1 and these IOP-associated genes in TM/SC tissue and the presence of 17ß-estradiol in AH supports a role for estrogen signaling in IOP regulation.

Autotaxin May Have Lysophosphatidic Acid-Unrelated Effects on Three-Dimension (3D) Cultured Human Trabecular Meshwork (HTM) Cells.

PURPOSE: The objective of the current study was to evaluate the effects of the autotaxin (ATX)-lysophosphatidic acid (LPA) signaling axis on the human trabecular meshwork (HTM) in two-dimensional (2D) and three-dimensional (3D) cultures of HTM cells. METHODS: The effects were characterized by transendothelial electrical resistance (TEER) and FITC-dextran permeability (2D), measurements of size and stiffness (3D), and the expression of several genes, including extracellular matrix (ECM) molecules, their modulators, and endoplasmic reticulum (ER) stress-related factors. RESULTS: A one-day exposure to 200 nM LPA induced significant down-sizing effects of the 3D HTM spheroids, and these effects were enhanced slightly on longer exposure. The TEER and FITC-dextran permeability data indicate that LPA induced an increase in the barrier function of the 2D HTM monolayers. A one-day exposure to a 2 mg/L solution of ATX also resulted in a significant decrease in the sizes of the 3D HTM spheroids, and an increase in stiffness was also observed. The gene expression of several ECMs, their regulators and ER-stress related factors by the 3D HTM spheroids were altered by both ATX and LPA, but in different manners. CONCLUSIONS: The findings presented herein suggest that ATX may have additional roles in the human TM, in addition to the ATX-LPA signaling axis.

Human trabecular meshwork cell behavior is influenced by collagen scaffold pore architecture and glycosaminoglycan composition.

Glaucoma is a degenerative eye disease in which damage to the optic nerve leads to a characteristic loss of vision. The primary risk factor for glaucoma is an increased intraocular pressure that is caused by an imbalance of aqueous humor generation and subsequent drainage through the trabecular meshwork (TM) drainage system. The small size, donor tissue limitations, and high complexity of the TM make it difficult to research the relationship between the TM cells and their immediate environment. Thus, a biomaterial-based approach may be more appropriate for research manipulations and in vitro drug development platforms. In this work, human TM (hTM) cells were cultured on various collagen scaffolds containing different glycosaminoglycans (GAGs) and different pore architectures to better understand how hTM cells respond to changes in their extracellular environment. Cellular response was measured by quantifying cellular proliferation and expression of an important extracellular matrix protein, fibronectin. The pore architecture of the scaffolds was altered using freeze-casting technique to make both large and small pores that were aligned or with a non-aligned random structure. The composition of the scaffolds was altered with the addition of chondroitin sulfate and/or hyaluronic acid. It was found that the hTM cells grown on large pore scaffolds proliferate more than those grown on small pores. There was an increase in the fibronectin expression with the incorporation of GAGs, and its morphology was changed by the underlying pore architecture. This work will help provide an insight into the behavior of hTM cells when introducing changes in their microenvironment.

Differentiation of microfluidic-encapsulated trabecular meshwork mesenchymal stem cells into insulin producing cells and their impact on diabetic rats.

Tissue and stem cell encapsulation andtransplantation were considered as promising tools in the treatment of patients with diabetes mellitus. The aim of this study was to evaluate the effect of microfluidic encapsulation on the differentiation of trabecular meshwork mesenchymal stem cells (TM-MSC), into insulin-producing cells (IPCs) both in vitro and in vivo. The presence of differentiated cells in microfibers (three dimensional [3D]) and tissue culture plates (TCPS; two dimensional [2D]) culture was evaluated by detecting mRNA and protein expression of pancreatic islet-specific markers as well as measuring insulin release of cells in response to glucose challenges. Finally, semi-differentiated cells in microfibers (3D) and 2D cultures were used to control the glucose level in diabetic rats. The results of this study showed that MSCs differentiated in alginate microfibers (fabricated by microfluidic device) express more Pdx-1 mRNA (1.938-fold, p-value: 0.0425) and Insulin mRNA (2.841-fold, p-value: 0.0001) compared with those cultured on TCPS. Furthermore, cell encapsulation in microfluidic derived microfibers decreased the level of blood glucose in diabetic rats. The approach used in this study showed the possibility of alginate microfibers as a matrix for differentiation of TM-MSCs (as a new source) into IPCs. In addition, it could minimize different steps in stem cell differentiation, handling, and encapsulation, which lead to loss of an unlimited number of cells.

The many faces of the trabecular meshwork cell.

With the combined purpose of facilitating useful vision over a lifetime, a number of ocular cells have evolved specialized features not found elsewhere in the body. The trabecular meshwork (TM) cell at the irido-corneal angle, which is a key regulator of intraocular pressure, is no exception. Examination of cells in culture isolated from the human TM has shown that they are unique in many ways, displaying characteristic features of several different cell types. Thus, these neural crest derived cells display expression patterns and behaviors typical of endothelia, fibroblasts, smooth muscle and macrophages, owing to the multiple roles and two distinct environments where they operate to maintain intraocular pressure homeostasis. In most individuals, TM cells function normally over a lifetime in the face of persistent stressors, including phagocytic, oxidative, mechanical and metabolic stress. Study of TM cells isolated from ocular hypertensive eyes has shown a compromised ability to perform their daily duties. This review highlights the many responsibilities of the TM cell and its challenges, progress in our understanding of TM biology over the past 30 years, as well as discusses unanswered questions about TM dysfunction that results in IOP dysregulation and glaucoma.

Collagen and collagen-chondroitin sulfate scaffolds with uniaxially aligned pores for the biomimetic, three dimensional culture of trabecular meshwork cells.

Glaucoma is a disease in which damage to the optic nerve leads to progressive, irreversible vision loss. The intraocular pressure (IOP) is the only modifiable risk factor for glaucoma and its lowering is considered a useful strategy for preventing or slowing down the progression of glaucomatous neuropathy. Elevated intraocular pressure associated with glaucoma is due to increased aqueous humor outflow resistance, primarily through the trabecular meshwork (TM) of the eye. Current in vitro models of the trabecular meshwork are oversimplified and do not capture the organized and complex three-dimensional nature of this tissue that consists primarily of collagen and glycoasaminoglycans. In this work, collagen and collagen-chondroitin sulfate (CS) scaffolds were fabricated via unidirectional freezing and lyophilization to induce the formation of aligned pores. Scaffolds were characterized by scanning electron microscopy, dynamic mechanical analysis, and a chondroitin sulfate quantification assay. Scaffold characterization confirmed the formation of aligned pores, and also that the CS was leaching out of the scaffolds over time. Primary porcine trabecular meshwork (TM) cells were seeded onto the surface of scaffolds and their gene expression, proliferation, viability, migration into the scaffolds, and morphology were examined. The TM cells were viable and proliferated 2 weeks after seeding. The cells migrated down into the internal scaffold structure and their morphology reflected the topography and alignment of the scaffold structure. This work is a promising step toward the development of a three dimensional in vitro model of the TM that can be used for testing of glaucoma pharmacological agents in future experimentation and to better our understanding of the trabecular meshwork and its complex physiology. Biotechnol. Bioeng. 2017;114: 915-923. © 2016 Wiley Periodicals, Inc.

The autophagic lysosomal system in outflow pathway physiology and pathophysiology.

Malfunction of the trabecular meshwork (TM)/schlemm's canal (SC) conventional outflow pathway is associated with elevated intraocular pressure (IOP) and, therefore, increased risk of developing glaucoma, a potentially blinding disease affecting more than 70 million people worldwide. This TM/SC tissue is subjected to different types of stress, including mechanical, oxidative, and phagocytic stress. Long-term exposure to these stresses is believed to lead to a progressive accumulation of damaged cellular and tissue structures causing permanent alterations in the tissue physiology, and contribute to the pathologic increase in aqueous humor (AH) outflow resistance. Autophagy is emerging as an essential cellular survival mechanism against a variety of stressors. In addition to performing basal functions, autophagy acts as a cellular survival pathway and represents an essential mechanism by which organisms can adapt to acute stress conditions and repair stress-induced damage. A decline in autophagy has been observed in most tissues with aging and has been considered responsible, at least in part, for the accumulation of damaged cellular components in almost all tissues of aging organisms. Dysfunction in the autophagy pathway is associated with several human diseases, from infectious diseases to cancer and neurodegeneration. In this review, we will summarize our current knowledge of the emerging roles of autophagy in outflow tissue physiology and pathophysiology, including novel evidence suggesting compromised autophagy in the glaucomatous outflow pathway.

Microstructure visualization of conventional outflow pathway and finite element modeling analysis of trabecular meshwork.

BACKGROUND: The intraocular pressure (IOP) is maintained through a dynamic equilibrium between the production and drainage of aqueous humor. Elevation of intraocular pressure is mainly caused by the blocking of aqueous humor outflow pathway. Therefore, it is particularly important to study the structure of drainage pathway and the effect of ocular hypertension at the process of aqueous humor outflow. METHODS: Conventional drainage pathway of aqueous humor, including trabecular meshwork (TM), Schlemm's canal (SC) and aqueous vein, were imaged by using trans-scleral imaging method with lateral resolution of 2 µm. For quantitative assessment, the morphological parameters of the TM were measured with different IOP levels via a combination of measurements and simulations. RESULTS: Images of the TM and the adjacent tissues were obtained. The porosity of TM with normal intraocular pressure varies from 0.63 to 0.74 as the depth increases, while in high IOP it is changed from 0.44 to 0.59. The diameter of aqueous vein varies from 32 to 43 µm, and is smaller than that of SC, which varies from 48 to 64.67 µm. CONCLUSIONS: Our research provides a non-contact method to visualize the microstructure of tissue for clinical examination associated with the blocking of the outflow pathway of aqueous humor in humans. The three-dimensional (3D) microstructures of limbus and the results of finite element modeling analysis of the TM model will serve for the future evaluation of new glaucoma surgical techniques.

[Role of microRNA in primary open-angle glaucoma].

The pathogenesis of primary open-angle glaucoma (POAG) is still unclear. The trabecular meshwork (TM) plays an important role for regulation of aqueous humor outflow. The alteration of morphology, structure of TM cells and expression of extracellular matrix (ECM) components contribute to the abnormal increase of outflow resistance leading to elevated intraocular pressure (IOP). Recently, it has been confirmed that a variety of microRNA (miRNA) such as miRNA-29, 24, 200c and so on were expressed in TM tissue and different miRNA can affect the TM function and the synthesis of ECM in different ways. In this paper, we will review the role of miRNA in POAG pathogenesis, in order to provide the basis for probing into pathogenesis of glaucoma. (Chin J Ophthalmol, 2016, 52: 476-480).

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.

Wnt inhibition induces persistent increases in intrinsic stiffness of human trabecular meshwork cells.

Wnt antagonism has been linked to glaucoma and intraocular pressure regulation, as has increased stiffness of human trabecular meshwork (HTM) tissue. We have shown culturing HTM cells on substrates that mimic the elevated stiffness of glaucomatous tissue leads to elevated expression of the Wnt antagonist secreted frizzled related protein 1 (SFRP1), suggesting a linkage between SFRP1 and HTM mechanobiology. In this study, we document biomechanical consequences of Wnt antagonism on HTM cells. Cells were treated with the Wnt antagonists (SFRP1, KY02111, and LGK-974) for 8 days and allowed to recover for 4 days. After recovery, intrinsic cell stiffness and activation of the Wnt pathway via ß-catenin staining and blotting were assayed. Basal cell stiffness values were 3.71 ± 0.37, 4.33 ± 3.07, and 3.07 ± kPa (median ± S.D.) for cells derived from 3 donors. Cell stiffness increased after 0.25 µg/mL (4.32 ± 5.12, 8.86 ± 8.51, 4.84 ± 3.15 kPa) and 0.5 µg/mL (16.75 ± 5.59, 13.18 ± 7.99, and 8.54 ± 5.77 kPa) SFRP1 treatment. Stiffening was observed after 10 µM KY02111 (10.72 ± 5.63 and 6.57 ± 5.53 kPa) as well as LGK-974 (9.60 ± 7.41 and 11.40 ± 9.24 kPa) treatment compared with controls (3.79 ± 1.01 and 5.16 ± 2.14 kPa). Additionally, Wnt inhibition resulted in decreased ß-catenin staining and increased phosphorylation at threonine 41 after recovery. In conclusion, this work demonstrates a causal relationship between Wnt inhibition and cell stiffening. Additionally, these findings suggest transient Wnt inhibition resulted in durable modulation of the mechanical phenotype of HTM cells. When placed in context with previous results, these findings provide a causal link between Wnt antagonism and cell stiffness and suggest a feedback loop contributing to glaucoma progression.

Mechanical analysis of rat trabecular meshwork.

Stiffness of trabecular meshwork (TM) may play an important role in regulating outflow resistance in healthy and glaucomatous eyes. However, the current techniques for stiffness measurement can only be applied to TM dissected from human donor or large animal eyes. It is a challenge to measure TM stiffness in mouse/rat eyes because of their smaller sizes and the delicate nature of TM dissection. To this end, a new technique was developed to determine the stiffness of rat TM using atomic force microscopy (AFM). In the study, rat eyes were enucleated immediately after death and perfused with a tracer (Evans blue) for 40 min. Then, the anterior segment was dissected and flat-mounted on a Petri dish with TM facing upwards. An AFM probe with a gold-coated colloid tip was used to sequentially indent the corneal, TM, and uveoscleral tissues. Assuming these tissues to be neo-Hookean materials, the indentation data were analyzed with a newly developed mathematical model to calculate the apparent initial Young's moduli (E0)(app). The geometric mean & SE of (E0)(app) were 162 Pa & 1.2 (n = 13) for TM and 6189 Pa & 1.4 (n = 11) for cornea; and the difference was statistically significant (p < 0.01). The technique established in this study allows the use of rat eye as a potential model for investigation of TM stiffness and its influences on outflow resistance. Future studies may also utilize this technique to evaluate mechanisms of TM stiffness change caused by aging, outflow dysfunction, pathogenesis of glaucoma, and drug treatment.

23-gauge transconjunctival vitrectomy in eyes with pre-existing functioning filtering blebs.

BACKGROUND: We investigated the outcome of 23-gauge transconjunctival pars plana vitrectomy (23G PPV) for the treatment of vitreoretinal disorder in patients with prior trabeculectomy. METHODS: We retrospectively reviewed medical records of 23G PPV in 11 eyes that had functioning filtering blebs after trabeculectomy. The main outcome measures were the visual acuity, intraocular pressure (IOP) and subconjunctival fluid height in bleb by anterior segment optical coherence tomography (OCT) before and after the surgery. RESULTS: Eyes that had 23G PPV showed improvement of visual acuity after the surgery (P =0.003). Mean IOP was 13.82 mmHg before 23G PPV and 15.82 mmHg at 6 months postoperatively, which was statistically insignificant (P = 0.758). The bleb was observed before and after surgery using anterior segment OCT, and the difference in subconjunctival fluid was not statistically significant (P =0.172). CONCLUSIONS: 23G PPV did not adversely affect bleb function in eyes with prior trabeculectomy.

Transplantation of mesenchymal stem cells promotes tissue regeneration in a glaucoma model through laser-induced paracrine factor secretion and progenitor cell recruitment.

Among bone marrow cells, hematopoietic and mesenchymal components can contribute to repair damaged organs. Such cells are usually used in acute diseases but few options are available for the treatment of chronic disorders. In this study, we have used a laser-induced model of open angle glaucoma (OAG) to evaluate the potential of bone marrow cell populations and the mechanisms involved in tissue repair. In addition, we investigated laser-induced tissue remodeling as a method of targeting effector cells into damaged tissues. We demonstrate that among bone marrow cells, mesenchymal stem cells (MSC) induce trabecular meshwork regeneration. MSC injection into the ocular anterior chamber leads to far more efficient decrease in intraocular pressure (IOP) (p < .001) and healing than hematopoietic cells. This robust effect was attributable to paracrine factors from stressed MSC, as injection of conditioned medium from MSC exposed to low but not to normal oxygen levels resulted in an immediate decrease in IOP. Moreover, MSC and their secreted factors induced reactivation of a progenitor cell pool found in the ciliary body and increased cellular proliferation. Proliferating cells were observed within the chamber angle for at least 1 month. Laser-induced remodeling was able to target MSC to damaged areas with ensuing specific increases in ocular progenitor cells. Thus, our results identify MSC and their secretum as crucial mediators of tissue repair in OAG through reactivation of local neural progenitors. In addition, laser treatment could represent an appealing strategy to promote MSC-mediated progenitor cell recruitment and tissue repair in chronic diseases.

Concentration-related effects of nitric oxide and endothelin-1 on human trabecular meshwork cell contractility.

The contractility status of trabecular meshwork (TM) cells influences aqueous humor outflow resistance and intraocular pressure. Using human TM cells as a model, the goal of the present study was to examine concentration-response relationships of two prototypical molecules, nitric oxide (NO) and endothelin-1 (ET-1), known to differentially influence vascular smooth muscle contractility. Efficacy of ET-1, two NO donors (DETA-NO and SNP) and a cGMP analog (8-Br-cGMP) were assessed using two complementary methods: functionally in a gel contraction assay and biochemically using a myosin light chain phosphorylation assay. The NO donors DETA-NO and SNP dose dependently relaxed cultured human TM cells (EC50 for DETA-NO = 6.0 ± 2.4 µM, SNP = 12.6 ± 8.8 µM), with maximum effects at 100 µM. Interestingly, at concentrations of NO donors above 100 µM, the relaxing effect was lost. Relaxation caused by DETA-NO (100 µM) was dose dependently blocked by the soluble guanylate cyclase specific inhibitor ODQ (IC50 = 460 ± 190 nM). In contrast to the NO donors, treatment of cells with the cGMP analog, 8-Br-cGMP produced the largest relaxation (109.4%) that persisted at high concentrations (EC50 = 110 ± 40 µM). ET-1 caused a dose-dependent contraction of human TM cells (EC50 = 1.5 ± 0.5 pM), with maximum effect at 100 pM (56.1%) and this contraction was reversed by DETA-NO (100 µM). Consistent with functional data, phosphorylation status of myosin light chain was dose dependently reduced with DETA-NO, and increased with ET-1. Together, data show that TM cells rapidly change their contractility status over a wide dynamic range, well suited for the regulation of outflow resistance and intraocular pressure.

The Factors Affecting the Stability of IOP Homeostasis.

Purpose: Shear-induced nitric oxide (NO) production by Schlemm's canal (SC) endothelial cells provides a fast, IOP-sensitive feedback signal that normally contributes to IOP homeostasis. Our goal was to analyze the response of this homeostatic system under constant flow perfusion (as occurs in vivo) vs. constant pressure perfusion (as typical for laboratory perfusions). Methods: A mathematical model of aqueous humor dynamics, including shear-mediated NO signaling, was formulated and analyzed for stability. The model includes Goldmann's equation, accounting for proximal and distal outflow resistance, and describes how elevated IOP causes narrowing of SC lumen that increases the shear stress on SC cells. Elevated shear stress stimulates NO production, which acts to reduce outflow resistance and relax trabecular meshwork cells to decrease trabecular meshwork stiffness, affecting the SC luminal caliber. Results: During constant flow perfusion, the outflow system is typically stable, returning to baseline IOP after a perturbation. In contrast, during constant pressure perfusion, the outflow system can become unstable and exhibit a time-dependent change in outflow resistance that diverges from baseline. Conclusions: The stability of shear mediated IOP homeostasis is predicted to differ critically between constant flow vs. constant pressure perfusion. Because outflow facility is typically measured at a constant pressure in the laboratory, this instability may contribute to the characteristic time-dependent increase in outflow facility, known as washout, observed in many nonhuman species. Studies of IOP homeostasis should consider how the outflow system may respond differently under constant pressure vs. constant flow perfusion.