Junhui Wu, Yalong Dang, Fang Lei
Purpose: To evaluate the ocular hypotensive effect of the Rho-associated kinase (ROCK) inhibitor RKI-1447 in normotensive and dexamethasone (DEX)-induced ocular hypertension rats, while elucidating its molecular mechanisms involving cytoskeletal remodeling and extracellular matrix (ECM) metabolism via the RhoA/ROCK pathway in trabecular meshwork (TM) cells. Methods: In vivo: Intraocular pressure (IOP) dynamics were monitored in normotensive and DEX-induced ocular hypertension rats following topical RKI-1447 administration. Conjunctival hyperemia, systemic safety (body weight), and IOP recovery post-withdrawal were assessed. In vitro: Primary human trabecular meshwork (HTM) cells were used to observe the effects of RKI-1447 on morphology and migration. Immunofluorescence assessed cross-linked actin networks (CLANs), ECM proteins, and alpha smooth muscle actin (α-SMA) expression. RhoA activity and the phosphorylation of myosin light chain (MLC) and myosin phosphatase target subunit-1 (MYPT-1) were detected via Pull-down assays and Western blot. Results: Topical RKI-1447 was associated with a dose-dependent trend in IOP reduction in both normotensive and DEX-induced ocular hypertension rats, with a peak effect observed at approximately 2 hours and only mild, transient hyperemia. In HTM cells, RKI-1447 treatment was observed to induce reversible morphological changes and mitigate cell migration. Furthermore, descriptive assessments suggested that RKI-1447 appeared to disassemble DEX-induced cytoskeletal assemblies and attenuate the upregulation of collage type IV (COL IV), fibronectin (FN), laminin (LN), and α-SMA. Additionally, RKI-1447 appeared to modulate DEX-associated RhoA activation and demonstrated a trend toward downregulating MLC and MYPT-1 phosphorylation. Conclusion: Our data suggest that RKI-1447 is associated with reduced aqueous outflow resistance, potentially through its inhibitory effect on the RhoA/ROCK pathway and the subsequent mitigation of steroid-induced cytoskeletal and fibrotic alterations. These preliminary observations suggest that RKI-1447 may serve as a viable pharmacological approach for treating steroid-induced glaucoma, although further long-term safety and efficacy evaluations are warranted.