Department of Ophthalmology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan.
Department of Ophthalmology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan.
Exp Eye Res. 2014 Jan;118:72-9. doi: 10.1016/j.exer.2013.11.014. Epub 2013 Nov 27.
The trabecular meshwork (TM) is a major component of the conventional outflow pathway and the excess extracellular matrix (ECM), and fibrosis in the TM causes increased outflow resistance. In this study, we first investigated the effects of several ECM components in the induction of an epithelial mesenchymal transition (EMT)-like phenomenon in TM cells. TM cells were isolated from cynomolgus monkeys (Macaca fascicularis). The cells were cultured in ECM-coated dishes and then subjected to both western blot analysis and immunocytochemistry to measure the levels of EMT-associated markers. Cell motility was assessed using wound healing and chemotaxis assays. We found that type I collagen, fibronectin and laminin induced the dissociation of cell-cell contact and elongation of actin stress fibers in the cultured monkey TM cells. In addition, following the same stimulation of the ECM, the expression of mesenchymal markers, such as fibronectin and α-smooth muscle actin, and the phosphorylation of Smad2 increased in the TM cells. Our results showed the significant acceleration of TM cellular motility following stimulation with type I collagen, fibronectin and laminin. These phenomena were inhibited by the c-Jun N-terminal kinase (JNK) inhibitor SP600125. In addition, siRNA against paxillin was transfected to evaluate the association between paxillin and the EMT-like phenomenon. The knockdown of paxillin expression by transfection with siRNA blocked the EMT-like alteration of the cellular characteristics and chemotaxis toward transforming growth factor-β2 in the cultured TM cells. Our results showed that the ECM-JNK-paxillin pathway induced an EMT-like phenomenon in TM cells, resulting in the abundant expression of fibronectin and activation of motility in TM cells. This EMT-like phenomenon could result in aberrant conditions in the aqueous outflow pathway in glaucomatous eyes.
小梁网（TM）是传统流出途径的主要组成部分，细胞外基质（ECM）的过度堆积和 TM 中的纤维化导致流出阻力增加。在这项研究中，我们首先研究了 ECM 成分对 TM 细胞上皮间质转化（EMT）样现象的诱导作用。我们从食蟹猴（Macaca fascicularis）中分离出 TM 细胞。将细胞培养在 ECM 包被的培养皿中，然后进行 Western blot 分析和免疫细胞化学分析，以测量 EMT 相关标志物的水平。使用划痕愈合和趋化性测定评估细胞迁移能力。我们发现 I 型胶原、纤维连接蛋白和层粘连蛋白诱导培养的猴 TM 细胞中细胞-细胞接触的解离和肌动蛋白应力纤维的伸长。此外，在相同的 ECM 刺激下，TM 细胞中间充质标志物（如纤维连接蛋白和α-平滑肌肌动蛋白）的表达以及 Smad2 的磷酸化增加。我们的结果表明，I 型胶原、纤维连接蛋白和层粘连蛋白刺激后 TM 细胞的迁移能力显著加快。这些现象被 c-Jun N 末端激酶（JNK）抑制剂 SP600125 抑制。此外，转染针对桩蛋白的 siRNA 以评估桩蛋白与 EMT 样现象之间的关联。转染 siRNA 敲低桩蛋白表达阻断了培养 TM 细胞中细胞特征的 EMT 样改变和向转化生长因子-β2 的趋化性。我们的结果表明，ECM-JNK-桩蛋白通路在 TM 细胞中诱导 EMT 样现象，导致纤维连接蛋白的大量表达和 TM 细胞的迁移能力激活。这种 EMT 样现象可能导致青光眼眼中水流出途径的异常。
