A new insight into the cellular regulation of aqueous outflow: how trabecular meshwork endothelial cells drive a mechanism that regulates the permeability of Schlemm's canal endothelial cells.

AIM: To test the hypothesis that trabecular meshwork endothelial cells (TMEs) increase the permeability of Schlemm's canal endothelial cells (SCEs) by actively releasing ligands that modulate the barrier properties of SCEs. METHODS: The TMEs were first irradiated with a laser light and allowed to condition the medium, which is then added to SCEs. The treatment response is determined by both measuring SCE permeability (flow meters) and the differential expression of genes (Affymetrix chips and quantitative polymerase chain reaction (PCR)). The cytokines secreted by the treated cells were identified using ELISA and the ability of these cytokines to increase permeability is tested directly after their addition to SCEs in perfusion experiments. RESULTS: SCEs exposed to medium conditioned by the light activated TMEs (TME-cm) respond by undergoing a differential expression (DE) of 1,120 genes relative to controls. This response is intense relative to a DE of only 12 genes in lasered SCEs. The TME-cm treatment of SCEs increased the SCE permeability fourfold. The role of cytokines in these responses is supported by two findings: adding specific cytokines established to be secreted by lasered TMEs to SCEs increases permeability; and inactivating the TME-cm by boiling or diluting, abrogates these conditioned media permeability effects. CONCLUSION: These experiments show that TMEs can regulate SCE permeability and that it is likely that TMEs have a major role in the regulation of aqueous outflow. This novel TME driven cellular mechanism has important implications for the pathogenesis of glaucoma and the mechanism of action of laser trabeculoplasty. Ligands identified as regulating SCE permeability have potential use for glaucoma therapy.

Glucocorticoids regulate transendothelial fluid flow resistance and formation of intercellular junctions.

The regulation of transendothelial fluid flow by glucocorticoids was studied in vitro with use of human endothelial cells cultured from Schlemm's canal (SCE) and the trabecular meshwork (TM) in conjunction with computer-linked flowmeters. After 2-7 wk of 500 nM dexamethasone (Dex) treatment, the following physiological, morphometric, and biochemical alterations were observed: a 3- to 5-fold increase in fluid flow resistance, a 2-fold increase in the representation of tight junctions, a 10- to 30-fold reduction in the mean area occupied by interendothelial "gaps" or preferential flow channels, and a 3- to 5-fold increase in the expression of the junction-associated protein ZO-1. The more resistive SCE cells expressed two isoforms of ZO-1; TM cells expressed only one. To investigate the role of ZO-1 in the aforementioned Dex effects, its expression was inhibited using antisense phosphorothioate oligonucleotides, and the response was compared with that observed with the use of sense and nonsense phosphorothioate oligonucleotides. Inhibition of ZO-1 expression abolished the Dex-induced increase in resistance and the accompanying alterations in cell junctions and gaps. These results support the hypothesis that intercellular junctions are necessary for the development and maintenance of transendothelial flow resistance in cultured SCE and TM cells and are likely involved in the mechanism of increased resistance associated with glucocorticoid exposure.

Effect of beta-adrenergic agonists on paracellular width and fluid flow across outflow pathway cells.

PURPOSE: To determine whether the adrenergic agonists epinephrine and isoproterenol regulate fluid flow across endothelial cells cultured from the human aqueous outflow pathway and to evaluate associated cellular mechanisms. METHODS: Confluent monolayers of human trabecular meshwork (TM) or Schlemm's canal endothelial (SCE) cells were grown on porous filter supports. The monolayers were perfused with media while fluid flow, expressed as hydraulic conductivity (HC = microl/min/mm Hg/cm2), was continuously measured in preparations treated with isoproterenol, epinephrine, or control medium. Morphometric ultrastructural methods were used to measure the area occupied by the intercellular space and by each cell. RESULTS: SCE cells and TM cells exposed to isoproterenol or epinephrine responded with an increase in transendothelial fluid flow. Dose-response curves for both adrenergic agonists showed that HC increased linearly as a function of the log of the isoproterenol and epinephrine concentration. At 10(-4) M isoproterenol, the HC increased threefold, and threshold conditions were reached at 10(-9) M. The increase in HC was apparent after isoproterenol had been applied for 1 hour, reached a peak in 3 to 4 hours, and declined gradually to return to baseline conditions in 10 to 12 hours. Morphometric analyses showed that the area occupied by the intercellular space increased fourfold when isoproterenol was used at 10(-4) M, whereas the cell area decreased as a function of the concentration of adrenergic agonist. Epinephrine's effects on HC and cell morphology were blocked by pretreatment with equimolar concentrations of the nonselective beta-blocker, timolol. CONCLUSIONS: Epinephrine and isoproterenol increase flow through the paracellular pathway of SCE and TM cells through a beta-receptor mediated response that widens the intercellular space and reduces cell area. These findings support the hypothesis that epinephrine decreases the intraocular pressure in glaucoma therapy by promoting fluid flow across the SCE and TM cells lining tissues of the major aqueous outflow pathway.

Epinephrine effects on major cell types of the aqueous outflow pathway: in vitro studies/clinical implications.

We have investigated the possibility that direct cellular effects may mediate the action of EPI to lower the IOP. To do this, cultured HTM and SCE cells were grown as monolayers over a millipore-filter support structure. The monolayers were exposed to various adrenergic agonists and antagonists while flow of the perfusate (DME + 5% FBS) was measured using a specially-designed computer-linked apparatus. Exposure of the cells to 10(-5) M EPI for around 2 hours led to a rapid twofold increase in HC which gradually declines over the next 12 hours. Continuous exposure of either cell type (ie, HTM or SCE cells) to EPI or ISO resulted in a four- to eightfold increase with a maximal effect measured around 10 hours and a half maximal effect at 2.5 hours. Administration of c-AMP alone gave similar responses. In agreement with clinical studies, timolol blocks EPI's effect completely while betaxolol acted as a partial antagonist. These findings suggest that the cellular changes and the increase in HC are mediated by a beta-2 receptor. There are many similarities between the responses observed using our in vitro system and the IOP-lowering response observed in vivo after the topical application of EPI (eg, concentration, time course, duration, and magnitude). The clinical implications of these preliminary results are discussed and it is proposed that the described in vitro system may be useful to select new adrenergic drugs for glaucoma therapy.

Kinetics of phagocytosis in trabecular meshwork cells. Flow cytometry and morphometry.

Confluent human trabecular meshwork (HTM) cells from three different donors and at various stages of serial passage were fed fluorescein-labeled polystyrene beads. Phagocytosis was monitored for up to 6 days using flow cytometry, fluorescence microscopy, and morphometric calculations from comprehensive electron microscopic observations at key time points. During the first 4 hr after initiation of phagocytosis, the confluent endothelial monolayer lost its cohesiveness and became segregated into separate cells. During the first 3 days the cells underwent marked and progressive changes in shape and size. After 4 days, some cells detached from the dish, as necrotic debris and degenerative changes appeared. The kinetics of phagocytosis in this stable, confluent monolayer showed that recruitment (the percentage of cells which had ingested at least one bead) proceeded semilogarithmically, with 50% of the cells recruited by 8 hr and 97% by 96 hr. The time course of phagocytosis (ie, the average number of beads phagocytosed per cell) is described by a sigmoidlike curve, reaching half-maximum at 40 hr and maximum (about 500 beads per cell) at 96 hr. The rate of uptake (ie, the first derivative of the average number of beads per cell) reached a peak (nine beads per cell per hr) at 24 hr and then decelerated slowly over the next 5 days. Cytochalasin B treatment, as a control, reduced phagocytosis by approximately 70%. Flow cytometry, when combined with electron microscopy, should provide a useful tool to examine phagocytosis in HTM cells exposed to steroids and other hormones and drugs.