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.

Prolonged use of dexamethasone (DEX) elevates intraocular pressure (IOP) and increases the risk of developing glaucoma. In a previous study, we demonstrate that DEX stimulates the expression of thrombospondin-1 (THBS1) in primary human trabecular meshwork (hTM) cells, and that inhibiting THBS1 expression prevents DEX-induced elevation of IOP in mice. Therefore, we investigate the mechanism by which DEX regulates THBS1 expression. Treatment with the DNA methylation inhibitors, 5-azacytosine (5-AC) or 5-aza-2’-deoxycytidine (5-aza-dC), upregulates THBS1 protein levels in vitro and in vivo, reduces outflow facility in perfused mouse eyes, and elevates IOP in mice. In primary hTM cells, 7-day DEX treatment results in hypomethylation of the THBS1 promoter region and reduces transcript levels of 2 DNA methyltransferases (DNMTs), DNMT1 and DNMT3A. Taken together, we show that DEX reduces expression of DNMTs and DNA methylation of the THBS1 promoter region, supporting a critical role for THBS1 in DEX-induced outflow reduction.