Department of Cell Biology, the Moorfields Eye Hospital/UCL Institute of Ophthalmology, London, United Kingdom.
Invest Ophthalmol Vis Sci. 2012 Jul 10;53(8):4682-91. doi: 10.1167/iovs.11-8577.
To evaluate the efficiency of Rac1 inhibition in preventing matrix contraction by Tenon's capsule fibroblasts.
The involvement of Rac1 in serum-stimulated matrix contraction by human Tenon's fibroblasts (HTFs) was investigated in a classic collagen contraction model and our ex vivo model of tissue contraction using immunocytochemistry, chemical inhibitors, and small interfering RNA (siRNA) technology. Matrix integrity was assessed using confocal reflection microscopy and Coomassie blue staining. Quantitative real-time polymerase chain reaction (QRT-PCR) and Western blot analysis were used to assess matrix metalloproteinase (MMP) expression.
Serum induced Rac1 activation in HTF-populated collagen gels and stimulated HTFs to contract collagen matrices down to ~90% of their original size. Rac1 inhibition using NSC23766 or depletion using siRNA both significantly reduced HTF-mediated contraction. Early brief exposure to NSC23766 reduced HTF-mediated gel contraction by 70%, while transient treatment with the Rac1 inhibitor once a week decreased ex vivo tissue contraction down to serum-free levels. Transient exposure to NSC23766 prevented early cell protrusions, fiber alignment, and matrix degradation, as seen upon continuous exposure to broad-spectrum MMP inhibitor. However, unlike MMP inhibition, transient treatment with NSC23766 led to a significant reduction in MMP1 mRNA and protein expression during contraction, without increasing MMP2 and MMP14 expression.
Rac1 inhibition efficiently prevents conjunctival tissue and collagen matrix contraction and prevents matrix degradation.
评估 Rac1 抑制在防止 Tenon 囊成纤维细胞基质收缩中的效率。
在经典的胶原收缩模型和我们的组织收缩离体模型中，通过免疫细胞化学、化学抑制剂和小干扰 RNA（siRNA）技术研究 Rac1 在人 Tenon 纤维（HTF）的血清刺激基质收缩中的作用。使用共聚焦反射显微镜和考马斯亮蓝染色评估基质完整性。使用定量实时聚合酶链反应（QRT-PCR）和 Western blot 分析评估基质金属蛋白酶（MMP）表达。
血清诱导 HTF 细胞填充的胶原凝胶中的 Rac1 激活，并刺激 HTF 收缩胶原基质，使其达到原始大小的~90%。使用 NSC23766 抑制 Rac1 或使用 siRNA 耗尽 Rac1 均显著减少了 HTF 介导的收缩。早期短暂暴露于 NSC23766 可使 HTF 介导的凝胶收缩减少 70%，而每周一次短暂用 Rac1 抑制剂处理可将离体组织收缩减少至无血清水平。短暂暴露于 NSC23766 可防止细胞突起、纤维排列和基质降解的早期出现，这与持续暴露于广谱 MMP 抑制剂相似。然而，与 MMP 抑制不同，短暂用 NSC23766 处理可导致收缩期间 MMP1 mRNA 和蛋白表达显著减少，而不会增加 MMP2 和 MMP14 表达。
Rac1 抑制可有效防止结膜组织和胶原基质收缩，并防止基质降解。
