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M. Zhang, R. Maddala, D.L. Epstein, P.V. Rao; Rho GTPase–Induced Trabecular Meshwork Cellular Contraction Causes a Decrease in Aqueous Humor Outflow Facility . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1868.
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Using constitutively active Rho GTPase, we determined the effects of increased trabecular meshwork contractile activity on aqueous humor outflow and on outflow pathway cell adhesions and junctions.
Adenoviral vectors expressing green fluorescent protein alone (Ad–GFP) or a constitutively active form of RhoA (V14) and GFP (Ad–RhoAV14–GFP) were utilized in this study. Human primary trabecular meshwork (TM) cells, Schlemm’s canal (SC) cells and porcine primary TM cells were infected with Ad–GFP alone or with Ad–RhoAV14–GFP. Changes in actin stress fibers, focal adhesions, adherens junctions, and the state of myosin light chain (MLC) phosphorylation were evaluated by immunofluorescence and immunoblotting methods. Aqueous humor outflow facility was determined in organ cultured anterior segments of porcine eyes infected with 5x107 pfu adenoviral vectors (Ad–GFP or Ad–RhoAV14–GFP) using a constant flow perfusion system.
Expression of a RhoAV14 mutant resulted in increased actin stress fiber (phalloidin), focal adhesion (vinculin) and adherens junction (beta–catenin) staining in both TM and SC cells. These cellular changes were associated with increased MLC phosphorylation and cell stiffness. Organ cultured porcine eye anterior segments expressing RhoAV14 and GFP exhibited a moderate but significant decrease in outflow facility (30%, n=6) compared to control anterior segments expressing GFP alone.
Taken collectively, these data indicate an association between RhoAV14–induced increases in the formation of cell adhesions, cell–cell junctions, and MLC phosphorylation which, in turn, could cause TM contraction and a decrease in aqueous outflow facility.
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