Abstract
Intercellular adherens junctions and cell-extracellular matrix interactions are presumed to influence aqueous humor (AH) drainage via the conventional route, however, their direct role in modulation of intraocular pressure (IOP) is not well understood. Here, we investigated the role of Rac GTPase signaling in basal and growth factor-induced formation of adherens junctions in human trabecular meshwork (HTM) cells as compared to human umbilical vascular endothelial cells, and evaluated the effects of inhibition of Rac GTPase activity on IOP in rabbits. Expression of a constitutively active Rac1 GTPase or treatment with platelet derived growth factor (PDGF), a known activator of Rac GTPase, induced formation of β-catenin-based adherens junctions, actin cytoskeletal reorganization and membrane ruffle in HTM cells. In contrast, treatment of HTM cells with inhibitors of Rac GTPase caused cell-cell separation, a decrease in adherens junctions, and reorganization of actin stress fibers to the cell cortical regions and focal adhesion to the cell leading edges. Both, constitutively active Rac1 and PDGF stimulated generation of Reactive Oxygen Species (ROS) in HTM cells, and ROS were found to increase adherens junction formation and transendothelial electrical resistance (TEER) in HTM cells. Topical application of Rac GTPase inhibitors (EHT1864 and NSC23766), however, only marginally influenced IOP in rabbit eyes. Taken together, these data reveal that while Rac GTPase signaling plays a significant role in regulation of adherens junctions, ROS production and TEER in cells of the AH outflow pathway, Rac inhibitors showed only a marginal influence on IOP in live rabbits.
Highlights
Elevated intraocular pressure (IOP) caused by increased resistance to aqueous humor (AH) outflow localized within the conventional/ trabecular pathway [1,2,3]
To understand the role of Rac GTPase signaling in regulation of actin cytoskeleton reorganization and adherens junctions formation in trabecular meshwork (TM) cells, we first determined the effects of Rac inhibitors on these processes
Confluent cultures of human trabecular meshwork (HTM) cells maintained overnight in 1% fetal bovine serum (FBS) were treated with Rac GTPase inhibitors- EHT1864 or NSC23766 (1 M to 50 mM; dissolved in phosphate buffered saline (PBS)) for 6 h
Summary
Elevated intraocular pressure (IOP) caused by increased resistance to aqueous humor (AH) outflow localized within the conventional/ trabecular pathway [1,2,3]. Other agents which are known to activate Rac GTPase activity and stimulate cell-cell junction formation including Sphingosine-1-phosphate, have been shown to decrease AH outflow facility in perfused model systems possibly via increased transendothelial permeability barrier of SC [7,10,31]. In these latter studies, the direct role of Rac GTPase activity in either cell-cell junction formation or on IOP has not been addressed. In this study, we evaluated the direct effects of Rac GTPase activation and inactivation on adherens junctions formation in HTM cells as compared to human umbilical vascular endothelial cells (HUVEC) used as a model SC endothelial cell type, and on IOP changes in a live animal model
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