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

Abstract

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.

Figure 1

Ratio intensity scatter plot comparing the profile of genes expressed by TMEs treated by the application of short pulse/green light delivered by a frequency doubled Nd:YAG laser instrument (A); by the addition of media conditioned by the irradiated TMEs (TME-cm) when added to naive TMEs (B); or by adding TME-cm to naive SCEs (C). Each dot represents the mean intensity log ratio (base 2) versus the average log intensity of a single gene based on four replicas (that is, one chip for each of four samples), with each replicate containing 11 probes/gene, using approximately 47 000 transcripts from 38 500 well characterised genes. Red and green dots indicate upregulated and downregulated genes, respectively, demonstrating a twofold or greater differential expression (DE) ratio with the total number of differentially expressed genes (DEGs) for each category indicated below each graph.

Figure 2

Ratio intensity scatter plot as in figure 1 showing profiles DEGs in SCEs after the lasering procedure (A); or after adding SCE-cm to naive SCEs (B); or after adding SCE-cm to naive TMEs (C).

Figure 3

(A) The conductivity responses in μl/min/mm Hg/cm², with the mean (SD), measured in monolayers of both TMEs and SCEs in controls and in treated preparations as indicated. In (B) the treatment responses are depicted adjusting for differences in baseline as a fold change. Note that in all cases, the responses are robust, amounting to increases of over 11-fold in the case of the TME-cm, the highest response, and 200% in the case of naive SCEs treated with SCE-cm, the smallest response.

Figure 4

Conductivity responses measured in μl/min/mm Hg/cm² showing the mean and SD in controls (blue) and in TMEs treated by the addition of media conditioned by light irradiated TMEs (red). Adding media from naive TMEs, or from laser activated TMEs that had been inactivated by dilution or boiling effectively abrogated the increase in conductivity (blue), relative to the situation when media conditioned by light irradiated TMEs was added (red).

Figure 5

Intensity plot depicting the mRNA responses (means (SD)) measured using quantitative PCR (Q-PCR) in the eight experimental and control preparations (A), and at the protein level measured using ELISA (B). These responses are correlated with those measured in conductivity assays (C). The mRNA responses are measured as the mean intensity log ratio (base 2). The ELISA measurements are expressed in pg/ml, and the conductivity studies units in μl/min/mm Hg/cm².

Figure 6

Plot showing changes in conductivity induced by the direct addition to SCE monolayers of 10 ng of IL1-α, 5 ng of IL-8, 15 ng of TNF-α, and 10 ng of IL-β?. Mean (SD) are shown.