Meyer-ter-Vehn Tobias, Sieprath Sonja, Katzenberger Barbara, Gebhardt Susanne, Grehn Franz, Schlunck Günther
Division of Experimental Ophthalmology and Glaucoma Center, Würzburg University Eye Hospital, Josef-Schneider-Strasse 11, D-97080 Würzburg, Germany.
Invest Ophthalmol Vis Sci. 2006 Nov;47(11):4895-904. doi: 10.1167/iovs.06-0118.
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.
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).
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.
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.
评估Rho激酶依赖性收缩性在转化生长因子-β（TGF-β）诱导人Tenon成纤维细胞向肌成纤维细胞转分化过程中的意义，以确定抑制青光眼手术后瘢痕形成的可能药理学靶点。
人Tenon成纤维细胞（HTFs）在培养中生长并用TGF-β1刺激。通过GST-罗激酶结合域下拉试验评估TGF-β对Rho-GTP酶活性的影响，并通过蛋白质印迹分析检测。在硅胶底物起皱试验和成纤维细胞填充的胶原凝胶中评估收缩性。通过免疫荧光显微镜观察肌动蛋白细胞骨架和粘着斑。通过实时逆转录聚合酶链反应测量α-平滑肌肌动蛋白（α-SMA）转录本。通过蛋白质印迹分析检测TGF-β诱导的Smad和p38激活以及α-SMA的表达。通过共聚焦免疫荧光显微镜确定Smad2/3的核转位。使用特异性激酶抑制剂（Y-27632、HA-1077、H-1152和ML-7）研究Rho依赖性激酶（ROCK）和肌球蛋白轻链激酶（MLCK）的影响。
在刺激后10分钟内，TGF-β诱导Rho激活，这与细胞张力增加相关，随后是肌动蛋白应力纤维增强。ROCK抑制剂释放细胞张力并避免TGF-β诱导的细胞骨架变化、p38激活和随后的α-SMA表达，而Smad2磷酸化和核转位得以保留。MLCK抑制也阻断α-SMA表达。在成纤维细胞填充的胶原晶格中，ROCK抑制剂阻止TGF-β诱导的应力纤维组装和收缩。
TGF-β在肌成纤维细胞转分化之前诱导HTFs快速收缩反应。ROCK抑制剂释放这种收缩并阻断随后TGF-β诱导的肌成纤维细胞转分化，因此可能有助于调节青光眼滤过手术后的瘢痕形成。
