Contractility as a prerequisite for TGF-β-induced myofibroblast transdifferentiation in human tenon fibroblasts

PURPOSE. To assess the significance of Rho-kinase-dependent contractility in TGF-beta-induced myofibroblast transdifferentiation of human tenon fibroblasts to characterize possible pharmacological targets for the inhibition of postoperative scarring after glaucoma surgery. METHODS. Human tenon fibroblasts (HTFs) were grown in culture and stimulated with TGF-beta 1. The effect of TGF-beta on Rho-GTPase activity was assessed by GST-rhotekin binding domain pulldown assay and detected by Western blot analysis. Contractility was evaluated in a silicone substrate wrinkling assay and in fibroblast-populated collagen gels. The actin cytoskeleton and focal adhesions were visualized by immunofluorescence microscopy. alpha-SMA transcripts were measured by real-time RT-PCR. TGF-beta-induced Smad- and p38-activation and expression of alpha-SMA were detected by Western blot analysis. Nuclear translocation of Smad2/3 was determined by confocal immunofluorescence microscopy. The influence of Rho-dependent kinase ( ROCK) and myosin light chain kinase (MLCK) were studied by using specific kinase inhibitors (Y-27632, HA-1077, H-1152, and ML-7). RESULTS. Within 10 minutes of stimulation, TGF-beta induced Rho activation that was associated with an increase in cell tension and followed by actin stress fiber enhancement. ROCK inhibitors released cell tension and averted TGF-beta-induced cytoskeletal changes, p38 activation and subsequent alpha-SMA expression, whereas Smad2-phosphorylation and nuclear translocation were preserved. MLCK inhibition also blocked alpha-SMA expression. In fibroblast-populated collagen lattices, ROCK inhibitors prevented TGF-beta-induced stress fiber assembly and contraction. CONCLUSIONS. TGF-beta induces a rapid contractile response in HTFs that precedes myofibroblast transdifferentiation. ROCK inhibitors release this contraction and block subsequent TGF-beta- induced myofibroblast transdifferentiation and may therefore serve to modulate postoperative scarring after glaucoma filtering surgery.