Abstract
Purpose :
Aqueous humor (AH) outflow and intraocular pressure (IOP) regulation are influenced by actin cytoskeletal organization and cell adhesive interactions. To deepen our understanding of their role in AH outflow and IOP regulation, this study investigates the impact of a Rho kinase inhibitor, a known ocular hypotensive agent, on the cytoskeletal and cell adhesive protein profile of human TM cells.
Methods :
Human TM cell strains from 41 and 71 year-old donors, along with their duplicates, were subjected to a 24-hour treatment with a Rho kinase inhibitor (Y27632, 10 µM). Subsequently, cytoskeletome fractions were extracted and analyzed using label-free quantitative proteomics with a Q Exactive HF mass spectrometer. Validation was performed through immunoblots, immunofluorescence, and RT-PCR analyses.
Results :
In the cytoskeletome fractions of Rho kinase inhibitor-treated TM cells, both decreased and increased levels of proteins were observed compared to controls. Y27632 treatment led to decreased levels of myosins, integrins, cell adhesion and junctional proteins, actin binding proteins, extracellular matrix components, galectins, MICAL2, septins, and glypicans. Surprisingly, several proteins exhibited significantly elevated levels in Y27632-treated TM cells compared to controls, including Matrix Remodeling-Associated Protein-7 (MXRA7), MXRA5, THSD4, serine protease (HTRA1), Copine-1, caveolin-1, TGFBI, TMX4, vitronectin, Thy1, and calpain-1. Notably, MXRA7 showed over a 25-fold increase in Y27632-treated cells, expressing significantly lower levels than MXRA5 and MXRA8 in normal TM cells. Importantly, the gene variant of MXRA5 has been shown to be associated with IOP and glaucoma in distinct cohorts.
Conclusions :
This study identifies differential protein levels in the cytoskeletome fraction of TM cells under Rho kinase inhibition. Notably, the Rho kinase inhibitor significantly elevates matrix remodeling-associated proteins (MXRA7 and MXRA5), offering novel insight on its mechanism of action for ocular hypotensive activity.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.