Effects of Dominant Negative Rho kinase Expression on the Contractile Properties of Trabecular Meshwork Cells and Aqueous Humor Outflow

Abstract

Abstract: : Purpose: Based on pharmacological inhibition of activity, Rho kinase has been proposed to modulate aqueous outflow and intraocular pressure (IOP). This study employs a molecular approach to determine the specific role of Rho kinase in modulation of aqueous humor outflow. Methods: Adenoviral vectors expressing green fluorescent protein alone (Ad–GFP) or dominant negative Rho kinase and GFP (Ad–DNRK–GFP) were utilized in these experiments. Human and porcine trabecular meshwork (TM) cells were infected at 30–60 MOI (multiplicity of infection) of Ad–GFP alone or with Ad–DNRK–GFP. Changes in cell shape, actomyosin cytoskeletal integrity and the status of myosin light chain phosphorylation were evaluated. Measurement of IOP and outflow facility was conducted using anterior chambers of human cadaver eyes infected with 1x10 ⁷ PFU adenoviral vectors (Ad–GFP or Ad–DNRK–GFP) using the organ culture constant flow system. Results: Overexpression of DNRK (confirmed by increased GFP fluorescence) resulted in changes of cell shape (cell rounding, cell–cell detachment) and loss of both actin stress fibers and focal adhesions in human and porcine TM cells. These cellular changes were associated with a dramatic decrease in myosin light chain phosphorylation. Additionally, in an ongoing study, a small number of cultured human eye anterior chambers (n=2) infected with Ad–GFP or Ad–DNRK–GFP exhibited a drop in IOP of 6% and 45%, respectively, compared to baseline values. Conclusions: This study demonstrates that specific inhibition of Rho kinase activity in trabecular meshwork cells alters the cell shape and contractile properties, and we hypothesize that these morphological events underlie the observed effects of Rho kinase on aqueous humor outflow.

Keywords: trabecular meshwork • cytoskeleton • outflow: trabecular meshwork