Lovastatin inhibits TGF-beta-induced myofibroblast transdifferentiation in human tenon fibroblasts.

PURPOSE: The transdifferentiation of Tenon fibroblasts to myofibroblasts is a pivotal step in filtering bleb scarring. It is mediated by the cytokine TGF-beta, Rho-dependent contractility, and cell-matrix interactions in an interdependent fashion. HMG-CoA-reductase inhibitors (statins) have been shown to inhibit Rho-GTPase signaling; therefore, the authors studied the influence of lovastatin on TGF-beta-mediated myofibroblast transdifferentiation to assess the potential use of statins in wound healing modulation. METHODS: Human Tenon fibroblasts were grown in culture, pretreated with lovastatin, lovastatin and mevalonate, or specific inhibitors of farnesyl transferase or geranylgeranyl transferase and were stimulated with TGF-beta1. alpha-Smooth muscle actin (alpha-SMA) and connective tissue growth factor (CTGF) transcription were assessed by real-time PCR. alpha-SMA protein expression and localization were studied by Western blot and confocal immunofluorescence microscopy. Cell contractility was determined in collagen gel contraction assays. Phosphorylation of the signaling proteins Smad-2/3 and p38 were detected by Western blot, and Smad-2/3 localization was determined by confocal immunofluorescence microscopy. RESULTS: Lovastatin inhibited TGF-beta-induced CTGF transcription, alpha-SMA expression and incorporation into actin stress fibers, and subsequent collagen gel contraction. These effects were reversed by mevalonate. The inhibition of geranylgeranyl transferase but not farnesyl transferase blocked TGF-beta-induced alpha-SMA expression. Lovastatin decreased TGF-beta-induced p38 activation, whereas Smad-2/3 phosphorylation and nuclear translocation were preserved. CONCLUSIONS: Lovastatin inhibits TGF-beta-induced myofibroblast transdifferentiation in human Tenon fibroblasts, most likely by interfering with Rho-signaling. Statins may, therefore, serve to inhibit scarring after filtering glaucoma surgery.

Contractility as a prerequisite for TGF-beta-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-beta1. 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.

Specification of dorsoventral polarity in the embryonic chick mesencephalon and its presumptive role in midbrain morphogenesis.

The chick midbrain is subdivided into functionally distinct ventral and dorsal domains, tegmentum and optic tectum. In the mature tectum, neurons are organized in layers, while they form discrete nuclei in the tegmentum. Dorsoventral (DV) specification of the early midbrain should thus play a crucial role for the organization of the neuronal circuitry in optic tectum and tegmentum. To investigate regional commitment and establishment of cellular differences along the midbrain DV axis, we examined the commitment of gene expression patterns in isolated ventral and dorsal tissue in vivo and in vitro, and studied their cell mixing properties. Use of explant cultures, and grafting of dorsal midbrain into a ventral environment or vice versa, revealed a gradual increase in the autonomy of region-specific gene regulation between stages 12 and 18 (embryonic day 2 to 3). This process becomes independent of the activity of midline organizers, such as floor and roof plate, by stage 16. Once the DV axis polarity is fixed, cells from dorsal and ventral midbrain adopt differential adhesive properties. Thus between stages 18 to 23 (embryonic day 3 and 4), cells of dorsal and ventral origin start to separate from each other, at a time-point when the majority of midbrain cells is not yet differentiated. Hence, our results suggest that progressive specification of the midbrain DV axis is accompanied by progressively reduced cell mixing between dorsal and ventral precursors, leading to a partial regionalization of midbrain tissue into autonomous units of precursor cell populations.