No abstract

No abstract

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.

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 1120 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.T he conventional aqueous outflow pathway (CAOP) performs the dual functions of facilitating the egress of aqueous from the anterior chamber of the eye into the lumen of Schlemm's canal and of preventing the reflux of blood from the venous circulation into the anterior chamber.

The purpose of this study was to compare human endothelial cells from Schlemm's canal (SCEs) and the trabecular meshwork (TMEs) in terms of ZO-1 isoform expression, hydraulic conductivity (HC) properties, and "giant" vacuole (GV) formation. The principal study methods were Western blot, RT-PCR, immunofluorescence, and perfusion chambers. Blot signals for alpha+ - and alpha- -isoforms were similar in SCEs but less intense for the alpha+ -relative to the alpha- -signal in TMEs. With the anti-alpha+ antibody used at 1/50 dilution, binding occurred at cell borders of both cell types, but only to SCEs when used at a >/=1/200 dilution in vitro and in vivo. SCEs were more resistive than TMEs (HC = 0.66 vs. 1.32 microl.min-1.mmHg-1.cm-2; P < 0.001) when perfused from apex to base. When perfused in the other direction, SCEs were again more resistive (5.23 vs. 9.04 microl.min-1.mmHg-1.cm-2; P < 0.01). GV formation occurred only in SCEs as a function of flow direction, perfusion pressure, and time. We conclude that SCEs and TMEs have distinctive phenotypic properties involving their content of ZO-1 isoforms, barrier function, and GV formation.

No abstract

To determine whether selective laser trabeculoplasty (SLT) induces monocyte recruitment to the trabecular meshwork (TM) in human and monkey eyes and whether monocytes increase both aqueous outflow in vivo and the conductivity of human Schlemm canal endothelial cells (SCEs) in vitro. Methods: Monocyte recruitment was examined morphometrically in control human and monkey eyes and compared with that following SLT applied 1 to 3 days earlier. Outflow facility was measured for up to 4 days after the intracameral infusion of autologous macrophages in rabbits. Schlemm canal endothelial cell conductivity was measured using flow meters after exposing cultured SCEs to monocytes and monocyte-secreted factors for 24 hours. Results: Our estimates show that the TM in the human eye normally had an average of 15 003 monocytes, while in the monkey eye there were 3181 monocytes, and this number increased 4-to 5-fold following SLT. The intra-cameral infusion of autologous macrophages in rabbits increased outflow facility 2-fold in a rapid and sustained manner. Human monocytes and monocytesecreted factors increased SCE conductivity 2-fold in vitro. Conclusions: The number of monocytes/macrophages in the TM increases substantially after SLT and monocytes augment both outflow facility and SCE conductivity. Clinical Relevance: These findings indicate that the innate immune system in general and monocytes in particular play an important role in aqueous outflow homeostasis. The recruitment of monocytes in increased numbers after SLT likely plays a role in lowering the intraocular pressure after this procedure. The intracameral introduction of autologous monocytes harvested from a vein could have therapeutic potential as a cell-based individualized treatment of glaucoma.

No abstract