Abstract
Purpose: :
The HepII domain and its putative 4β1 integrin binding sequence PPRARI increase outflow facility (OF) in monkey organ-cultured anterior segments (MOCAS). The aim of this study was to determine if HepII signaling through 4β1 integrins decreases contractility in trabecular meshwork (TM) cells and how it affects the organization of the TM in MOCAS.
Methods: :
TM cells were transfected with 100 nM siRNA targeting 4 or a nontargeting control and grown to confluence. Cells were incubated with or without 500 µg/ml HepII for 24 hrs. Levels of 4β1 integrin expression were measured by FACS using the anti-4β1 antibody P1H4 and changes in actin cytoskeleton were determined by immunofluorescence using Alexa 488-phalloidin. The role of Rac1 and RhoA was determined by analyzing TM lysates treated with or without 250 µg/ml of HepII for 24 hrs with G-LisaTMRac and Rho activation kits. To assess contractility, TM cells were grown on 1.25 mg/ml rat tail collagen I gels for 24 hrs and incubated with or without 250 µg/ml of HepII for 24 hrs. Gels were detached from the dishes and their diameters were measured. OF was determined by two-level constant pressure perfusion in anterior segments of rhesus and cynomolgus monkey eyes. One segment from each pair was exchanged with 100 µg/ml of HepII, while the other was exchanged with DMEM. MOCAS were embedded in epon 812 and semi-thin sections were stained with toluidine blue for light microscopy.
Results: :
4β1 integrin expression was decreased by 89% in siRNA transfected TM cells. Untransfected cells treated with HepII appeared rounded and lacked actin filaments, while transfected cells treated with HepII did not differ in appearance from untreated control cells. The HepII domain did not alter the activity of Rac1, but it decreased the activity of RhoA by 30%. TM cells treated with HepII decreased collagen I gel contraction by nearly 50% compared to untreated controls. HepII increased OF in 2 out of 3 MOCAS by 52±19% after an overnight infusion. Light microscopy showed that MOCAS perfused with the HepII domain had an expansion of the space between the inner wall of Schlemm's canal and the trabecular collagen beams.
Conclusions: :
The HepII domain signals through 4β1 integrins. Activation of this signaling pathway downregulates RhoA activity and decreases the contractility of TM cells. The effect of HepII on MOCAS appeared similar to that previously observed with Lat-B. These data suggest that the HepII domain may be using an 4β1 integrin signaling pathway to increase OF and that the increase in OF may be the result of a decrease in the contractile properties of the TM.
Keywords: trabecular meshwork • cytoskeleton • signal transduction