Effects of chemical inhibition of N-WASP, a critical regulator of actin polymerization on aqueous humor outflow through the conventional pathway.
Effects of chemical inhibition of N-WASP, a critical regulator of actin polymerization on aqueous humor outflow through the conventional pathway.
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DOI：
10.1016/j.exer.2009.11.015
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发表时间：
2010-02
影响因子：
3.4
通讯作者：
Rao, P. Vasantha
中科院分区：
文献类型：
--
作者：
Inoue, Toshihiro;Pattabiraman, Padmanabhan P.;Epstein, David L.;Rao, P. Vasantha
在房水流出通路中，肌动蛋白细胞骨架组织的完整性被认为在调节通过小梁网的房水流出中起关键作用。然而，我们对流出通路中肌动蛋白细胞骨架动力学的调节的理解非常有限。为了探讨神经Wiskott-Aldrich综合征蛋白（N-WASP）在房水流出通路中的潜在重要性，采用猪眼球摘除和恒压灌流系统评价了选择性抑制剂Wiskostatin对房水流出功能的影响。此外，通过免疫荧光法、酶谱法和质谱法测定了药物对猪小梁网（TM）细胞中肌动蛋白细胞骨架组织、细胞粘附、肌球蛋白II磷酸化、基质金属蛋白酶（MMP）活性和细胞骨架蛋白谱的影响。在Wiskostatin灌注的猪眼中，房水流出功能显著且进行性增加。Wiskostatin灌注的眼睛似乎表现出水丛内壁中的巨大空泡增加和水丛变形。Wiskostatin处理的TM细胞表现出广泛的空泡在其胞质溶胶中，肌动蛋白应力纤维和局灶性粘连以可逆的方式减少。药物处理的TM细胞也显示减少肌球蛋白II和肌动蛋白的细胞骨架富集的Triton不溶性部分，但不影响肌球蛋白II磷酸化或MMP-2的活性。这些数据表明，N-WASP的化学抑制增加了与减少的肌动球蛋白相互作用和细胞粘附相互作用相关的房水流出设施，揭示了N-WASP在房水流出的稳态中的重要性。
The integrity of actin cytoskeletal organization in aqueous humor outflow pathway is thought to play a critical role in modulation of aqueous humor outflow through the trabecular meshwork. Our understanding of the regulation of actin cytoskeletal dynamics in outflow pathway, however, is very limited. To explore the potential importance of Neural Wiskott-Aldrich syndrome protein (N-WASP), a critical regulator of actin polymerization/nucleation in aqueous humor outflow pathway, the effects of Wiskostatin, a selective pharmacological inhibitor of N-WASP, on aqueous humor outflow facility were evaluated using enucleated porcine eyes and a constant pressure perfusion system. Further, drug induced effects on actin cytoskeletal organization, cell adhesions, myosin II phosphorylation, matrix metalloproteinase (MMP) activity, and cytoskeletal protein profile in porcine trabecular meshwork (TM) cells were determined by immunofluorescence, zymography, and mass spectrometry. Aqueous humor outflow facility was increased significantly and progressively in the Wiskostatin perfused porcine eyes. The Wiskostatin perfused eyes appear to exhibit increased giant vacuoles in the inner wall of aqueous plexi and deformation of aqueous plexi. The Wiskostatin treated TM cells demonstrated extensive vacuoles in their cytosol, and both actin stress fibers and focal adhesions were decreased in a reversible manner. The drug treated TM cells also revealed decreased myosin II and actin in the cytoskeletal enriched triton insoluble fraction but did not affect myosin II phosphorylation or MMP-2 activity. These data demonstrate that the chemical inhibition of N-WASP increases aqueous humor outflow facility in association with decreased actomyosin interaction and cell adhesive interactions revealing the importance of N-WASP in homeostasis of aqueous humor outflow.
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