Novel Molecular Insights Into Rho GTPase-Induced Resistance to Aqueous Humor Outflow Through the Trabecular Meshwork

Abstract

Purpose:: To gain insight into the molecular basis for the RhoA GTPase-induced increase in resistance to aqueous outflow through the conventional outflow pathway, we analyzed the effects of expressing constitutively active RhoA GTPase in cultured human trabecular meshwork (TM) cells on genome-wide gene expression profiles.

Methods:: The effects of RhoA activation on aqueous outflow facility were measured using organ cultured porcine eye anterior segments expressing an adenovirally encoded constitutively active RhoA (RhoA V14). Total RNA extracted from HTM cells expressing RhoAV14 was subjected to cDNA microarray using arrays representing ≈36,000 transcripts and quantitative PCR analyses.

Results:: Expression of RhoAV14 in organ cultured porcine eye anterior segments for one week led to a marginal decrease (≈ 10%) in outflow facility relative to the baseline. Under similar conditions, control eyes expressing the green fluorescent protein (GFP) demonstrated a 23 % increase in aqueous outflow facility. Outflow facility values were significantly different between the RhoAV14 and GFP expressing eyes, starting from 36 hours post infection and continuing till 100 hours. Primary HTM cells expressing RhoAV14 exhibited increased actin stress fibers, focal adhesions and adherens junctions compared to GFP expressing controls. cDNA microarray analysis of RNA extracted from RhoAV14 expressing HTM cells revealed a significant increase in the expression of genes encoding extracellular matrix (ECM) proteins (perlecan, brevican, myocilin, collagen, fibronectin and laminin), cytokines (IL-1ß, TGF-beta2, compliment factor H1, CTGF and TNF-5), cytoskeletal proteins (talin, ZO-3, caldesmon, integrins and filamin A) and signaling proteins (Ca²⁺ ATPase, Wnt-11, cGMP-dependent phosphodiestarase 5A2, cAMP-phosphodiesterase 4A, PI3 kinase, IP3 kinase, and myosin light chain kinase) compared to GFP expressing controls.

Conclusions:: Abnormal activation of Rho GTPase signaling appears to not only influence contractile properties of TM cells and cell adhesive interactions, but notably also the expression of cytokines, ECM proteins and cyclic nucleotide hydrolyzing phosphodiesterases which might potentially increase aqueous humor outflow resistance through the trabecular pathway.

Keywords: outflow: trabecular meshwork • gene/expression • signal transduction