Choi-Ying Ling

Rho-associated protein kinase (ROCK) inhibitors reduce cytoskeletal tension in trabecular meshwork (TM) cells, but the mechanisms linking this acute response to sustained extracellular matrix remodelling remain incompletely understood. In this study, we investigated whether the ROCK inhibitor Y39983 regulates thrombospondin-1 (THBS1) and DNA methylation programs, elements known to modulate extracellular matrix (ECM) remodelling, in primary human trabecular meshwork (hTM) cells. Primary hTM cells were treated with Y39983, 5-azacytidine (5-Aza, a DNA methyltransferase inhibitor), or both in combination. Gene expression of THBS1, DNA methyltransferase, ECM, and cytoskeletal molecules was assessed using RT-qPCR. THBS1 promoter methylation was examined through methylation-specific PCR, while genome-wide methylation remodelling was profiled using whole-genome bisulphite sequencing (WGBS) followed by pathway enrichment analysis. 5-Azacytidine abolished Y39983-dependent effects on DNMT1 and DNMT3A and attenuated THBS1 regulation. Methylation-specific PCR suggested altered THBS1 promoter methylation after Y39983 treatment, while whole-genome bisulphite sequencing identified 8845 differentially methylated genes, including 5608 hypomethylated and 3237 hypermethylated genes, enriched in ErbB signalling, focal adhesion, and regulation of the actin cytoskeleton. Independently, Y39983 reduced the basal and stimulus-induced expression of THBS1, as well as ECM- and cytoskeleton-associated genes, including COL4A1, COL6A1, and ACTG1. These findings demonstrate that ROCK inhibition is associated with changes in DNMT-related gene expression and DNA methylation patterns in hTM cells. However, because 5-azacytidine has effects beyond selective DNMT inhibition, further studies using orthogonal approaches are required to establish whether DNA methylation directly mediates transcriptional responses to Y-39983.

Age-related macular degeneration (AMD) is a degenerative eye disease leading to central vision loss and is characterized by dysregulated autophagy of the retinal pigment epithelium (RPE) layer. Recent studies have suggested that rho-associated protein kinase (ROCK) inhibitors may enhance autophagy in neurodegenerative diseases and promote the survival of RPE cells. This study investigated the effect of ROCK inhibitors on autophagy gene expression and autophagic vacuole formation in a human RPE (ARPE-19) cell line. The highly selective and potent ROCK inhibitor Y-39983 enhanced the expression of autophagy genes in ARPE-19 cells and increased autophagic vacuole formation. A proteomic analysis using mass spectrometry was performed to further characterize the effects of ROCK inhibition at the protein level. Y-39983 downregulated thrombospondin-1 (THBS1), and suppression of THBS1 in ARPE-19 cells resulted in an increase in autophagic vacuole formation. Our data showed that ROCK inhibitor-induced autophagy was mediated by THBS1 downregulation. We identified ROCK and THBS1 as potential novel therapeutic targets in AMD.

Secretion of melatonin, a natural hormone whose receptors are present in the ciliary epithelium, displays diurnal variation in the aqueous humor (AH), potentially contributing to the regulation of intraocular pressure. This study aimed to determine the effects of melatonin on AH secretion in porcine ciliary epithelium. The addition of 100 µM melatonin to both sides of the epithelium significantly increased the short-circuit current (Isc) by ~40%. Stromal administration alone had no effect on the Isc, but aqueous application triggered a 40% increase in Isc, similar to that of bilateral application without additive effect. Pre-treatment with niflumic acid abolished melatonin-induced Isc stimulation. More importantly, melatonin stimulated the fluid secretion across the intact ciliary epithelium by ~80% and elicited a sustained increase (~50–60%) in gap junctional permeability between pigmented ciliary epithelial (PE) cells and non-pigmented ciliary epithelial (NPE) cells. The expression of MT₃ receptor was found to be >10-fold higher than that of MT₁ and MT₂ in porcine ciliary epithelium. Aqueous pre-treatment with MT₁/MT₂ antagonist luzindole failed to inhibit the melatonin-induced Isc response, while MT₃ antagonist prazosin pre-treatment abolished the Isc stimulation. We conclude that melatonin facilitates Cl⁻ and fluid movement from PE to NPE cells, thereby stimulating AH secretion via NPE-cell MT₃ receptors.