Optimizing gene transfer to conventional outflow cells in living mouse eyes.

The mouse eye has physiological and genetic advantages to study conventional outflow function. However, its small size and shallow anterior chamber presents technical challenges to efficient intracameral delivery of genetic material to conventional outflow cells. The goal of this study was to optimize methods to overcome this technical hurdle, without damaging ocular structures or compromising outflow function. Gene targeting was monitored by immunofluorescence microscopy after transduction of adenovirus encoding green fluorescent protein driven by a CMV promoter. Guided by a micromanipulator and stereomicroscope, virus was delivered intracamerally to anesthetized mice by bolus injection using a 33 gauge needle attached to Hamilton syringe or infusion with glass micropipette connected to syringe pump. The total number of particles introduced remained constant, while volume of injected virus solution (3-10 µl) was varied for each method and time of infusion (3-40 min) tested. Outflow facility and intraocular pressure were monitored invasively using established techniques. Unlike bolus injections or slow infusions, introduction of virus intracamerally during rapid infusions (3 min) at any volume tested preferentially targeted trabecular meshwork and Schlemm's canal cells, with minimal transduction of neighboring cells. While infusions resulted in transient intraocular pressure spikes (commensurate with volume infused, Δ40-70 mmHg), eyes typically recovered within 60 min. Transduced eyes displayed normal outflow facility and tissue morphology 3-6 days after infusions. Taken together, fast infusion of virus solution in small volumes intracamerally is a novel and effective method to selectively deliver agents to conventional outflow cells in living mice.

Protein markers and differentiation in culture for Schlemm's canal endothelial cells.

The two cell types that populate the human conventional outflow pathway, Schlemm's canal (SC) and trabecular meshwork (TM) regulate intraocular pressure. In culture, SC and TM cells have been useful tools toward understanding their respective roles in conventional outflow homeostasis. Unfortunately, currently available protein markers that distinguish SC from TM cells are limited, motivating the present study. Antibodies that specifically recognize different vascular endothelial markers were used to probe lysates from mature cell monolayers subjected to SDS-PAGE followed by western blot analyses. Results show that SC and TM cells both expressed many of the endothelial candidate proteins investigated, such as Robo1/4, Tie2/TEK, VEGF-R1/R2, VCAM-1, eNOS and neuropilin-1. In contrast, all SC cell strains tested (n=11) expressed two proteins, fibulin-2 and vascular endothelial (VE) cadherin, not expressed by TM cells. To examine changes in VE-cadherin expression and cell-cell junction formation, indicated by transendothelial electrical resistance (TEER), SC cells were seeded onto filters at confluence and growth factors were withdrawn. Culturing cells in media containing adult bovine serum rather than fetal bovine serum resulted in a 75% mean increase in TEER and 67% corresponding average increase in VE-cadherin expression (p<0.05). While both TM and SC cells form monolayers, are contact inhibited, share some endothelial responsibilities and several endothelial protein markers, SC cells uniquely express at least two proteins which likely reflect a distinction in cellular responsibilities in vivo. One of these responsibilities, maintenance of the blood-aqueous barrier, can be modeled in culture upon withdrawal of growth factors from SC cell monolayers.

Role of aquaporin-1 in trabecular meshwork cell homeostasis during mechanical strain.

Aquaporin-1 (AQP1) channels are expressed by trabecular meshwork (TM) and Schlemm's canal cells of the conventional outflow pathway where fluid movement is predominantly paracellular, suggesting a non-canonical role for AQP1. We hypothesized that AQP1 functions to protect TM cells during periods of mechanical strain. To test this idea, primary cultures of confluent human TM cells on Bioflex membranes were exposed to static and cyclic stretch for 8 and 24h using the Flexcell system. AQP1 expression in TM cells was assessed by SDS-PAGE and Western blot using anti-AQP1 IgGs. AQP1 protein bands were analyzed using densitometry and normalized to beta-actin expression. Cell damage was monitored by measuring lactate dehydrogenase (LDH) and histone deacetylase appearance in conditioned media. Recombinant expression of AQP1 in TM cell cultures was facilitated by transduction with adenovirus. Results show that AQP1 expression significantly increased 2-fold with 10% static stretch and 3.5-fold with 20% static stretch at 8h (n=4, p<0.05) and 24h (n=6, p<0.05). While histone deacetylase levels were unaffected by treatments, release of LDH from TM cells was the most profound at the 20% static stretch level (n=4, p<0.05). Significantly, cells were refractory to the 20% static stretch level when AQP1 expression was increased to near tissue levels. Analysis of LDH release with respect to AQP1 expression revealed an inverse linear relationship (r(2)=0.7780). Taken together, AQP1 in human TM appears to serve a protective role by facilitating improved cell viability during conditions of mechanical strain.

Forskolin stimulation of water and cation permeability in aquaporin 1 water channels.

Aquaporin 1, a six-transmembrane domain protein, is a water channel present in many fluid-secreting and -absorbing cells. In Xenopus oocytes injected with aquaporin 1 complementary RNA, the application of forskolin or cyclic 8-bromo- adenosine 3',5'-monophosphate increased membrane permeability to water and triggered a cationic conductance. The cationic conductance was also induced by direct injection of protein kinase A (PKA) catalytic subunit, reduced by the kinase inhibitor H7, and blocked by HgCl2, an inhibitor of aquaporin 1. The cationic permeability of the aquaporin 1 channel is activated by a cyclic adenosine monophosphate-dependent mechanism that may involve direct or indirect phosphorylation by PKA.

A biomimetic Schlemm's canal inner wall: A model to study outflow physiology, glaucoma pathology and high-throughput drug screening.

Glaucoma is a disease that damages the optic nerve, frequently leading to blindness. Elevated intraocular pressure (IOP) is the only modifiable risk factor for glaucoma, which is expected to affect 80 million people by 2020, causing bilateral blindness in over 10 million individuals. Because pathological changes to Schlemm's canal (SC) may account for significant resistance to outflow, there is considerable interest in characterizing and evaluating the Schlemm's canal as a target for glaucoma therapeutics. In conventional, two-dimensional culture, human Schlemm's canal (HSC) cells lose spatial, mechanical and biochemical cues, resulting in altered gene expression and cell signaling than observed in vivo, compromising the clinical relevance of data obtained from such systems. Here, we report, for the first time, that 3D culture of HSC cells on microfabricated scaffolds with defined physical and biochemical cues, rescued expression of key HSC markers, VE-cadherin and PECAM1, and mediated pore formation, crucial for the Schlemm's canal regulation of IOP. We demonstrated that following treatment with the glaucopathogenic agent, TGF-ß2, HSC cells undergo an endothelial-mesenchymal transition, which together with the increase in extracellular matrix (ECM) proteins might account for the decrease in outflow facility observed in patients with high TGF-ß2 levels in their aqueous humor. We also demonstrated that unlike 2D cultures, 3D cultures of HSC cells are amenable to gene transfer. Thus, our data imply that 3D culture of HSC cells may be used as a platform to advance our understanding of HSC physiology and pathology and as a model for high-throughput drug and gene screening.

Hydraulic pressure stimulates adenosine 3',5'-cyclic monophosphate accumulation in endothelial cells from Schlemm's canal.

PURPOSE: Fluid flow across various endothelia results in a variety of intracellular and extracellular adaptations. In the living eye, aqueous humor flows across the surface of endothelial cells on trabecular meshwork (TM) beams and in the juxtacanalicular tissue and through or between a continuous monolayer of endothelial cells that line Schlemm's canal (SC). This study was undertaken to test the hypothesis that fluid flow induces biochemical changes in the endothelial cells of the outflow pathway that may modify outflow resistance. METHODS: Trabecular meshwork and SC cells isolated from the outflow pathway of human cadaveric eyes were seeded onto porous filters, placed in Ussing-type chambers, and subjected to fluid flow driven by a pressure head of 15 mm Hg on their apical surface. Cell lysates were prepared and analyzed for adenosine 3',5'-cyclic monophosphate (cAMP) accumulation. Barrier function of cell monolayers was examined using transendothelial electrical resistance measurements. RESULTS: Three different SC cell strains in 14 independent experiments responded with at least a threefold increase in cAMP that was both time and pressure dependent. Conversely, flow-treated TM cells failed to respond in six independent experiments in which five different TM cell strains were used. Electrical resistance across cell monolayers positively correlated with cAMP accumulation and was calcium sensitive. CONCLUSIONS: cAMP signaling is affected by pressure differentials across SC cell monolayers and provides evidence for the participation of SC cells in the regulation of aqueous outflow.

Effect of AQP1 expression level on Co(2) permeability in bovine corneal endothelium.

PURPOSE: Corneal endothelial fluid transport is dependent on HCO(3)(-) and CO(2) fluxes. CO(2) permeability (P:CO(2)) measurements in an oocyte expression system and in reconstituted proteoliposomes have suggested that the water channel AQP1 can transport CO(2). An AQP1 knockout mouse model, however, showed no evidence for CO(2) transport through AQP1 in erythrocytes or lung. Because HCO(3)(-) and CO(2) fluxes are essential to endothelial function, the current study was conducted to determine whether AQP1 expression levels in confluent cultures of bovine corneal endothelial cells (BCECs) affects membrane PCO(2). METHODS: BCEC endogenous AQP1 expression was reduced by antisense oligonucleotide (AO) transfection or adenoviral antisense-AQP1 (AV) infection. AQP1 was overexpressed by adenoviral sense-AQP1 (SV) infection, which directs expression of recombinant AQP1. RESULTS: Expression of AQP1 and osmotic water permeability (control P(f) = 0.046 +/- 0.005 cm/sec) were reduced 45% and 36.5%, respectively, by AO transfection and reduced 67% and 49%, respectively, by AV infection. SV infection induced a more than threefold overexpression of AQP1 but showed only a 37% increase in P(f). Adenoviral empty virus (EV) infection did not change AQP1 expression or P(f). PCO(2) was determined by measuring the rate of intracellular pH decrease after exposure to CO(2)/HCO(3)(-)-rich solutions, as measured by the pH-sensitive fluorescent dye 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). Apparent PCO(2) of BCEC (0.0036 +/- 0.00023 cm/sec) was not different among control, oligonucleotide-transfected, and adenoviral-infected cells. P(f) could also be reduced more than 50% by 3 to 5 minutes' exposure of control cells to 0.5 mM p-chloromercuriphenylsulfonic acid (pCMBS), but this had no effect on rates of intracellular pH decrease. CONCLUSIONS: AQP1 does not contribute to PCO(2) in corneal endothelial cells.

Localization of alpha 2-adrenergic receptor subtypes in the anterior segment of the human eye with selective antibodies.

PURPOSE: To develop antibodies that selectively recognize each of the alpha 2-adrenergic receptor (AR) subtypes and to determine the expression and localization of these subtypes in the anterior segment of the human eye. METHODS: Recent studies have shown that there are three subtypes of the alpha 2-ARs, termed alpha 2-C10 (alpha 2A), alpha 2-C2 (alpha 2B), and alpha 2-C4 (alpha 2C). Polymerase chain reaction was used to amplify portions of these receptors fused (in-frame) to a cDNA encoding glutathione-S-transferase (GST). The expressed fusion proteins were used to immunize chickens, and antibodies were generated. Immunofluorescence microscopy was used to localize the alpha 2-AR subtypes in sections of human and rabbit ciliary body. Polymerase chain reaction and dot blot hybridization were used to determine which subtypes were present in RNA from primary cultures of human nonpigmented epithelium (NPE) and rabbit iris-ciliary body (ICB). RESULTS: Immunofluorescence microscopy of COS cells transfected with the alpha 2-AR subtypes showed that the antibodies raised against the GST-receptor fusion proteins specifically recognized their respective receptor subtypes. In the human ciliary body, alpha 2 B and alpha 2C immunoreactivity were present in the NPE and ciliary muscle. In the rabbit ciliary body, alpha 2A immunoreactivity also was present. Polymerase chain reaction and dot blot hybridization indicated that RNA encoding the alpha 2B and alpha 2C subtypes was present in human NPE and that RNA encoding all three subtypes was present in the rabbit ICB. CONCLUSIONS: Multiple alpha 2-adrenergic subtypes are expressed in the ciliary body. In the human, alpha 2B and alpha 2C predominate, whereas all three are present in the rabbit. This could be important with respect to animal models of glaucoma and to the development of drugs for lowering intraocular pressure.

Localization of aquaporin CHIP in the human eye: implications in the pathogenesis of glaucoma and other disorders of ocular fluid balance.

PURPOSE: The existence of integral membrane proteins that serve as selective water channels has been postulated to explain the movement of water across plasma membranes. Aquaporin CHIP (channel-forming integral membrane protein of 28 kd) is the first such channel to be characterized and is abundant in human erythrocytes and a variety of secretory and absorptive epithelia of the rat. Because disturbances in the movement of water characterize several ocular diseases, the distribution of CHIP in the human eye was studied. METHODS: Affinity-purified antibodies against purified CHIP protein were used for the indirect immunofluorescence localization of CHIP in human eye structures. Labeling was confirmed by immunoblot analyses of membrane preparations from eye structures. RESULTS: CHIP immunolabeling was found in the corneal endothelium, the lens epithelium, the nonpigmented epithelium of the ciliary process, the iris epithelium, and the endothelium of the trabecular meshwork and the canal of Schlemm. CONCLUSIONS: The presence of CHIP water channels in the secretory and absorptive tissues of the human eye provides a mechanism for transcellular water movement and may be important for understanding diseases of the eye that involve excess or insufficient movement of ocular fluid such as glaucoma, cataracts, and Fuch's dystrophy. In addition, the existence of CHIP in the outflow pathways of the human eye provides a novel explanation for the movement of water out of the eye.

Prostaglandin F2 alpha receptors in the human trabecular meshwork.

PURPOSE: Prostaglandin F2 alpha (PGF2 alpha) and analogs, such as latanoprost, are thought to lower intraocular pressure (IOP), primarily by increasing uveoscleral outflow. However, outflow through the trabecular meshwork may be increased as well. The authors hypothesize that any effect on the trabecular meshwork is mediated by prostanoid FP receptors (receptors for prostaglandin F2 alpha) in this tissue. METHODS: To test this hypothesis, tissue sections of the human trabecular meshwork and cultures of human trabecular meshwork cells were examined for the presence of FP receptors using immunofluorescence microscopy with affinity-purified antibodies raised against a glutathione-S-transferase (GST)-FPA receptor fusion protein. The presence of the receptor was confirmed by using reverse transcription-polymerase chain reaction (RT-PCR), functional assays of PGF2 alpha-stimulated inositol phosphate hydrolysis, and intracellular calcium measurements. RESULTS: Positive FPA receptor immunolabeling was observed in sections of the human trabecular meshwork and in cultured human trabecular meshwork cells. In both cases, specific labeling could be blocked by preincubation with a GST-FPA receptor fusion protein. Cross-blocking experiments with other receptor fusion proteins did not block specific labeling in cultured trabecular meshwork cells. PGF2 alpha caused a dose-dependent increase in total inositol phosphate accumulation and intracellular calcium release in human trabecular meshwork cells that was consistent with the presence of FP receptors. Using RT-PCR, message-encoding prostanoid FPA receptors were found in total RNA isolated from human trabecular meshwork cells. CONCLUSIONS: Prostanoid FPA receptors exist in human trabecular meshwork cells, as shown by the presence of mRNA, protein, and functional response to PGF2 alpha. This study indicates that functional FP receptors are present in the human trabecular meshwork and that they may be involved in mediating some of the IOP-lowering effects of PGF2 alpha in the eye.

Isolation, culture, and characterization of endothelial cells from Schlemm's canal.

PURPOSE: An important goal in glaucoma research has been to understand the functional contribution of trabecular meshwork (TM) and Schlemm's canal (SC) endothelia to aqueous humor outflow resistance. To date, TM cells are routinely cultured and used as a model by several laboratories. However, there has been only limited success in isolating SC cells. The current objective was to develop a technique for selective isolation and culture of endothelial cells from human SC. METHODS: The anterior chamber of human cadaveric eyes was cut into eight equal and radially symmetric wedge-shaped pieces. Using a dissecting microscope, a gelatin-coated suture (6-0 sterile nylon monofilament) was gently inserted into the lumen of SC and advanced into the canal. The cannulated pieces of tissue were placed in culture medium and maintained for 3 weeks. Sutures were removed from SC and cells seeded onto 3-cm culture plates. Morphology, growth characteristics, and expression of endothelial surface antigens and other cellular markers were evaluated. RESULTS: Of the 20 pairs of eyes that were cannulated, primary cells were obtained from 13. All SC cell isolates had a fusiform morphology; formed nonoverlapping, linearly arranged monolayers; and were contact inhibited. Schlemm's canal cell isolates reacted with antibodies specific for CD44 (hyaluron receptor), CD54 (intercellular adhesion molecule-1, ICAM-1), tissue-type plasminogen activator, and TM-inducible glucocorticoid-responsive protein-myocilin (TIGR-MYOC). Unlike TM cells, however, TIGR-MYOC protein was not induced in SC cells after long-term dexamethasone treatment. Schlemm's canal cells endocytosed low-density lipoprotein and acetylated low-density lipoprotein, and in the presence of Matrigel organized into multicellular tubelike structures. CONCLUSIONS: Cannulation of SC with gelatin-coated suture material is an effective method for the isolation of human SC cells and provides a cellular model to study the potential role of SC cells in aqueous humor outflow function.

Cultured human trabecular meshwork cells express functional alpha 2A adrenergic receptors.

PURPOSE: For the treatment of glaucoma, alpha-2 adrenergic receptor (alpha 2-AR) agonists are thought to lower intraocular pressure primarily by decreasing aqueous humor production. Effects on the outflow pathways, however, also may occur. To begin to examine this possibility, the authors characterized the alpha 2-AR subtypes present in cultures of human trabecular meshwork (HTM) cells using both immunofluorescence microscopy and functional measures of alpha 2-AR activation. METHODS: For immunofluorescence microscopy, subtype-specific polyclonal antibodies that recognize each of the human alpha 2-AR subtypes (alpha 2A, alpha 2B, alpha 2C) were used. Functional studies involved the inhibition of forskolin-stimulated cyclic adenosine monophosphate (cAMP) production, the stimulation of mitogen-activated protein (MAP) kinase activity, and the stimulation of mitotic activity as reflected by the expression of proliferating cell nuclear antigen (PCNA). RESULTS: From the immunofluorescence microscopy, there was evidence for the presence of the alpha 2A subtype, but not alpha 2B or alpha 2C subtype, on HTM cells. The administration of the alpha 2-agonist, dexmedetomidine, to HTM cells resulted in a 90% inhibition of forskolin-stimulated cAMP formation, a twofold stimulation of MAP kinase activity, and a threefold increase in the expression of PCNA. Additionally, preincubation of cells with either of the alpha 2-AR-selective antagonists, rauwolscine or atipamezole, reversed the functional effects of dexmedetomidine. CONCLUSIONS: Functional alpha 2A-ARs are present on HTM cells where they may affect the outflow pathway during the treatment of glaucoma with alpha 2-AR agonists.

Expression of aquaporin-1 in human trabecular meshwork cells: role in resting cell volume.

PURPOSE: Drainage of aqueous humor from the human eye appears dependent on intracellular volume of trabecular meshwork (TM) cells, the predominant cell type of the human outflow pathway. Thus, the modulation of water and solute flux across the plasma membrane of TM cells is predicted to be an important factor in regulating outflow facility. Aquaporin (AQP)-1 is a hexahelical integral membrane protein that functions as a regulated channel for water and cations in fluid-secreting and -absorbing tissues. AQP1 is present in many tissues of the human eye, including the TM; however, its role in outflow facility is unknown. The purpose of the present study was twofold: to evaluate the prospect of manipulating AQP1 protein levels in TM cells using sense and antisense mRNA and to investigate the functional role of AQP1 in TM cells. METHODS: An adenovirus (AV) expression system was used to alter AQP1 protein levels. AQP1 protein expression was monitored using immunoblot analysis, and resting cell volume was measured by forward light scatter, electronic cell sizing, and [(14)C]-sucrose/urea equilibration. Permeability of TM monolayers to [(14)C]-sucrose was also assessed as an indirect evaluation of cell volume. RESULTS: AV-mediated gene transfer of AQP1 cDNA to TM cells resulted in a titer-dependent increase in recombinant AQP1, whereas transfer of antisense cDNA decreased native AQP1 protein by 71.7% +/- 5.5% (P < 0.01) after 5 days. A novel finding of this study is that mean resting volumes of AQP1(s) AV-infected TM cells in suspension were 8.7% +/- 3.0% greater (P < 0.05) than control cells. Conversely, AQP1 antisense (as) AV-infected cells had resting volumes 7.8% +/- 2.9% less than control cells (P < 0.05). Similar effects of AQP1 expression on resting cell volume were observed in TM monolayers. Consistent with this finding, paracellular permeability of AQP1(s) AV-infected TM monolayers to [(14)C]-sucrose decreased by 8.0% +/- 1.4% (P < 0.001). CONCLUSIONS: In addition to influencing the osmotic permeability of TM plasma membranes, the level of AQP1 protein expression influences resting intracellular volume and thus paracellular permeability of TM cell monolayers in vitro. These data suggest that AQP1 expression may affect outflow facility in vivo.

Cannabinoid CB(1) receptor expression, activation and detection of endogenous ligand in trabecular meshwork and ciliary process tissues.

Elevated intraocular pressure is the primary risk factor for glaucoma. Cannabinoids interact with molecular targets in the eye and lower intraocular pressure by an unknown mechanism. The purpose of the present study was to examine eye tissues for functional cannabinoid receptors of the neuronal, CB(1) class, and an endogenous ligand, anandamide. The trabecular meshwork and ciliary processes are the primary structures of the eye that contribute to intraocular pressure and thus were our focus. Total RNA, frozen sections, cellular membranes and primary cultures of cells were prepared from both bovine and cadaveric human tissues. Using cannabinoid CB(1) receptor-specific oligodeoxynucleotide primers, cannabinoid CB(1) receptor antiserum, and cannabinoid-specific compounds (CP-55,940, WIN55,212-2 and SR-141716A), the presence of cannabinoid CB(1) receptors in ciliary processes and trabecular meshwork was determined. Using reverse transcription-polymerase chain reaction, we identified mRNA encoding cannabinoid CB(1) receptor protein in ciliary process and trabecular meshwork cells. Specific binding of anti-CB(1) immunoglobulin-G in tissue sections localized cannabinoid CB(1) receptor protein to the non-pigmented epithelial cells of the ciliary process and cells of the trabecular meshwork. While CP-55,940 and WIN55,212-2 failed to stimulate [(35)S]GTP gamma S binding in membrane preparations from trabecular meshwork and ciliary process, CP-55,940 significantly stimulated whole cell [(35)S]GTP gamma S binding by 51% over basal in ciliary process epithelial cells and 69% over basal in trabecular meshwork cells permeabilized with 5 microM digitonin (p<0.001). Specificity of agonist stimulation was verified by complete blockade with the specific cannabinoid CB(1) receptor antagonist, SR-141716A. Moreover, activation of cannabinoid CB(1) receptors by CP-55,940 resulted in a 2.3+/-0.3 and 1.7+/-0.3-fold stimulation of cAMP accumulation in trabecular meshwork and ciliary process cells, respectively (p<0.01). Lastly, anandamide was detected in human trabecular meshwork (3.08 pmol/g), ciliary process (49.42 pmol/g) and neurosensory retinal (4.48 pmol/g) tissues. These data, for the first time, demonstrate in a single study the presence of both CB(1) mRNA and protein in trabecular meshwork and ciliary processes from two different species. Activation of heterotrimeric G-proteins and stimulation of cAMP accumulation by cannabinoids in vitro suggest that their intraocular pressure-lowering effects in vivo result from activation of cannabinoid CB(1) receptors in the trabecular meshwork and increase aqueous outflow.

Dexamethasone decreases tissue plasminogen activator activity in trabecular meshwork organ and cell cultures.

Previously, we observed that dexamethasone decreased the extracellular activity of some matrix metalloproteinases and tissue plasminogen activator in trabecular meshwork containing organ explants and cell cultures. We have extended these studies to determine whether the decrease in extracellular tissue plasminogen activator activity is the result of a decrease in the expression of tissue plasminogen activator, a change in the extracellular level of plasminogen activator inhibitor-1, or a combination of both. We demonstrate that dexamethasone decreases the steady-state levels of tissue plasminogen activator mRNA by 75% in trabecular meshwork cells using Northern blot analysis, and show that this occurs concurrently with a 31% increase in the levels of extracellular plasminogen activator inhibitor-1 as determined by enzyme-linked immunosorbent assay analysis. A 38% increase was also measured in the extracellular plasminogen activator inhibitor-1 levels in trabecular meshwork containing organ explants after treatment. The amount of active, extracellular tissue plasminogen activator decreased by 72% in trabecular meshwork organ explant cultures, and 96% in trabecular meshwork cell cultures after treatment as determined using a parabolic rate assay for tissue plasminogen activator activity. These data suggest that dexamethasone decreases the extracellular activity of tissue plasminogen activator in trabecular meshwork containing organ and cell cultures by decreasing the expression of tissue plasminogen activator and increasing the extracellular levels of plasminogen activator inhibitor-1.

Effect of hydrostatic pressure gradients and Na2EDTA on permeability of human Schlemm's canal cell monolayers.

PURPOSE: Elevated intraocular pressure in those with glaucoma appears to be a function of increased resistance to movement of aqueous humor through the conventional outflow pathway. The majority of resistance in both normal and glaucomatous eyes is generated in the region between the juxtacanalicular trabecular meshwork and the inner wall of Schlemm's canal. To accommodate transient elevations in pressure, we hypothesize that conventional outflow increases rapidly due to changes in complexity of intercellular junctions between cells of the inner wall of Schlemm's canal. METHODS: To test this hypothesis we examined specifically the effects of hydrostatic pressure gradients and the calcium chelator, Na2EDTA, on permeability of cultured human Schlemm's canal cell monolayers in isolation. Human Schlemm's Canal cells were isolated, cultured and then seeded onto permeable supports and maintained in culture to allow intercellular junctions to mature. With a minimum net transendothelial electrical resistance of 10 Ohm cm2, cells were placed into an Ussing-type chamber and hydraulic conductivity was calculated from pressure and flow measurements that were continuously recorded. Simultaneously, transendothelial electrical resistance was measured manually at fixed intervals. In parallel experiments, cell margins were monitored in real time by videomicroscopy. RESULTS: During the baseline measurement period when cells were exposed to pressure but not Na2EDTA, hydraulic conductivity was constant but transendothelial electrical resistance decreased continuously at rate of 0.24 Ohm cm2/minute. After Na2EDTA treatment, no significant change in transendothelial electrical resistance was measured while, hydraulic conductivity of Schlemm's Canal monolayers increased significantly by 125%; corresponding to noticeable intercellular separations. Restoration of cell-cell contact was observed by videomicroscopy 30 minutes following washout of Na2EDTA and functionally after 2 hours. CONCLUSIONS: Responses of Schlemm's Canal cells to pressure and calcium chelators in vitro are consistent with a role for calcium sensitive junctions in outflow resistance in vivo.

Isolation and culture of human trabecular meshwork cells by extracellular matrix digestion.

Like corneal endothelial cells, human trabecular meshwork cells are believed to be of neural crest origin, but demonstrate physiological properties and an antithrombogenic surface similar to vascular endothelial cells. One current method for isolating trabecular meshwork cells utilizes the motile nature of these cells to migrate away from a trabecular meshwork explant in culture to more distal regions of the culture dish. This 'outgrowth' technique is limited in practice by the relatively small number of cells that migrate per explant per unit time, thus hindering the ability to gather sufficient numbers of cells for comprehensive experimentation. For this reason, we have modified an extracellular matrix digestion technique in current use for the isolation of microvascular endothelial cells to isolate human trabecular meshwork cells. This procedure is both efficient and rapid for isolating large numbers of trabecular meshwork cells and results in the availability of trabecular meshwork cells in sufficient quantities for subsequent experimentation.

Cultured human trabecular meshwork cells express aquaporin-1 water channels.

The identification and characterization of aquaporin-1 water channels and other related proteins has provided a molecular explanation for the enhanced permeability of a variety of epithelial tissues. Previously, we documented the distribution of aquaporin-1 in the human eye, which included the trabecular meshwork; the primary outflow channel for aqueous humor. The goal of this study was to determine if aquaporin-1 could be detected in cultures of human trabecular meshwork cells. Using primers specific for aquaporin-1, reverse transcription combined with polymerase chain reaction yielded a product of the appropriate size with total RNA prepared from the human trabecular meshwork cells. The presence of this product and its size (298 base pairs), is consistent with the presence of an aquaporin-1 message in these cells. Indirect immunofluorescence microscopy with affinity purified antibodies against a fusion protein containing the carboxy tail of aquaporin-1 showed specific labeling of the plasma membrane and immunoblotting identified a band of Mr 28,000 which agrees with the molecular size of aquaporin-1. The presence of aquaporin-1 in human trabecular meshwork cells, the predominant cell-type of the primary outflow region of the human eye, suggests that water channels may be involved with the movement of aqueous fluid out of the eye. In addition, the existence of aquaporin-1 on cultures of human trabecular meshwork cells provides an in vitro model to study the endogenous expression of aquaporin-1 and its possible role in the regulation of aqueous outflow.

Mechanical responsiveness of the endothelial cell of Schlemm's canal: scope, variability and its potential role in controlling aqueous humour outflow.

Primary open-angle glaucoma is associated with elevated intraocular pressure, which in turn is believed to result from impaired outflow of aqueous humour. Aqueous humour outflow passes mainly through the trabecular meshwork (TM) and then through pores formed in the endothelium of Schlemm's canal (SC), which experiences a basal-to-apical pressure gradient. This gradient dramatically deforms the SC endothelial cell and potentially contributes to the formation of those pores. However, mechanical properties of the SC cell are poorly defined. Using optical magnetic twisting cytometry and traction force microscopy, here we characterize the mechanical properties of primary cultures of the human SC cell, and for the first time, the scope of their changes in response to pharmacological agents that are known to modulate outflow resistance. Lysophosphatidic acid, sphingosine-1-phosphate (S1P) and thrombin caused an increase in cell stiffness by up to 200 per cent, whereas in most cell strains, exposure to latrunculin A, isoproterenol, dibutryl cyclic-AMP or Y-27632 caused a decrease in cell stiffness by up to 80 per cent, highlighting that SC cells possess a remarkably wide contractile scope. Drug responses were variable across donors. S1P, for example, caused 200 per cent stiffening in one donor strain but only 20 per cent stiffening in another. Isoproterenol caused dose-dependent softening in three donor strains but little or no response in two others, a finding mirrored by changes in traction forces and consistent with the level of expression of ß(2)-adrenergic receptors. Despite donor variability, those drugs that typically increase outflow resistance systematically caused cell stiffness to increase, while in most cases, those drugs that typically decrease outflow resistance caused cell stiffness to decrease. These findings establish the endothelial cell of SC as a reactive but variable mechanical component of the aqueous humour outflow pathway. Although the mechanism and locus of increased outflow resistance remain unclear, these data suggest the SC endothelial cell to be a modulator of outflow resistance.