Effects of novel ethacrynic acid derivatives on human trabecular meshwork cell shape, actin cytoskeletal organization, and transcellular fluid flow.

To determine efficacy and therapeutic index in the context of ocular hypotensive activity of the new ethacrynic acid (ECA) derivatives of the series (SA8,248 and SA8,389), 9,000 series (SA9,000, SA9,622 and SA9,995) and ticrynafen, we undertook a comparative evaluation of the dose-dependent effects of these compounds on human trabecular meshwork (HTM) cell shape, actin cytoskeletal organization, focal adhesions and transcellular fluid flow. Responses were either scored using an arbitrary scale of 1-5 or quantified. Compounds of the 9000 series (SA9,995>SA9,000>SA9,622) were found to be 14- to 20-fold more potent than ECA, ticrynafen or analogs from the 8,000 series (SA8,389>SA8,248) in terms of ability to induce cell shape alterations in HTM cells. Similarly, compounds of the 9,000 series (SA9,995>SA9,622>SA9,000) were found to be much stronger (2 to 20 fold) than ECA, ticrynafen or analogs of the 8000 series in terms of affecting decreases in actin stress fiber content in HTM cells. Analogs of the 9000 series (SA9,622>SA9,995>SA9,000) were also observed to be 8 to 10 fold more potent than ECA (SA8,389>ECA>SA8,248>ticrynafen) at eliciting decreases in cellular focal adhesions. Interestingly, analogs of the 9000 series (SA9,000>SA9,622>SA9,995) and SA8,248 demonstrated a huge increase (by many folds) in transcellular fluid flow of HTM cell monolayers as compared to ECA and ticrynafen. Collectively, these analyses revealed that the structural modification of ECA improves its ocular hypotensive efficacy, indicating that the SA9,000 series compounds might be promising novel ocular hypotensive drugs.

Interactions between endothelia of the trabecular meshwork and of Schlemm's canal: a new insight into the regulation of aqueous outflow in the eye.

PURPOSE: To test the hypothesis that trabecular meshwork endothelial cells (TMEs) regulate aqueous outflow by actively releasing ligands that upon binding to Schlemm's canal endothelial cells (SCEs) increase transendothelial flow, thereby facilitating the egress of aqueous. METHODS: We tested our hypothesis by (1) activating the TMEs in vitro using a laser procedure known to increase aqueous outflow in vivo; (2) demonstrating that lasered TMEs become activated at the genome-wide level and synthesize ligands; (3) ascertaining that media conditioned by laser-activated TMEs and ligands therein increase transendothelial flow when added to SCEs; and (4) determining that ligands identified as synthesized by TMEs increase permeability when added to SCEs. RESULTS: We find that adding either media conditioned by lasered TMEs or ligands synthesized by TMEs to naïve control SCEs increases permeability. Adding media boiled, diluted, or conditioned by nonlasered TMEs abrogates these permeability effects. Media conditioned by either lasered TMEs or SCEs (TME-cm/SCE-cm), when added to untreated controls of each cell type, induce congruous gene expression and flow effects: TME-cm induces far more differentially expressed genes (829 in control TMEs and 1,120 in control SCEs) than does the SCE-cm (12 in control TMEs and 328 in control SCEs), and TME-cm also increases flow much more (more than 11-fold in control TMEs and more than fourfold in control SCEs) than does the SCE-cm (fivefold in control TMEs and twofold in control SCEs). CONCLUSIONS: As postulated, the TMEs release factors that regulate SCE permeability. Derangement of this TME-driven process may play an important role in the pathogenesis of glaucoma. Ligands identified, which regulate permeability, have potential use for glaucoma therapy.

Endothelia of Schlemm's canal and trabecular meshwork: distinct molecular, functional, and anatomic features.

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.