Effects of long-term application of topical ROCK inhibitor on corneal endothelium and Descemet’s membrane of TAZ deficient mice

Abstract

Purpose : The Wwtr1 gene encodes the transcriptional co-activator with PDZ-binding motif (TAZ), which is a mechanotransducer that can be activated via Rho GTPase activity and actin cytoskeletal tension. Mice deficient in TAZ demonstrate a softer Descemet’s membrane (DM) and decreased corneal endothelial cell (CEnC) density. The purpose of this study was to evaluate the impact of Rho-associated protein kinase (ROCK) inhibition on CEnCs and DM in wildtype (WT) and heterozygous TAZ (Wwtr1^+/-) knockout mice.

Methods : Beginning at 6 months of age, 12 WT and 12 Wwtr1^+/- mice were treated with topical netarsudil 0.02% ophthalmic solution (Rhopressa^©) or vehicle control twice daily for 6 months. Endothelial cell density (ECD) was monitored using in vivo confocal microscopy. To compare the degree of age-related CEnC loss over time, % ECD change from baseline values were calculated. Following 6 months of treatment, all mice were euthanized and eyes were collected. For the right eyes, elastic modulus of Descemet’s membrane (DM) was measured with atomic force microscopy. The left eyes were used for immunofluorescence staining of actin-phalloidin on corneal endothelial wholemounts.

Results : The Wwtr1^+/- mice had softer DM than WT mice in control groups; however, treatment with topical netarsudil significantly stiffened DM of Wwtr1^+/- mice compared with treated control (Wwtr1^+/+) mice. There were no statistical differences in % ECD change between netarsudil-treated versus control groups at each timepoint in both WT and Wwtr1^+/- mice. While ECD was mildly lower in Wwtr1^+/- versus WT mice at baseline, no differences were observed between groups at study termination. Distinct actin-phalloidin staining pattern such as bleb-like appearance was observed in netarsudil-treated groups of both WT and Wwtr1^+/- mice consistent with cytoskeletal rearrangement by topical ROCK inhibition.

Conclusions : Topical netarsudil increased DM stiffness in Wwtr1^+/- mice to approximate that of WT control mice, suggesting that ROCK inhibition modulates CEnC-matrix interactions. The lack of ECD change is likely due to the mild phenotype displayed by this cohort of Wwtr1^+/- mice. Further study with Wwtr1^-/- mice with more severe ECD loss is warranted to determine the impact of long-term ROCK inhibition on CEnC loss.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.