Role of Rho Kinase in Aqueous Humor Outflow Pathway

Rho 激酶在房水流出途径中的作用

• 批准号: 6790613
• 负责人: P VASANTHA Rao
• 金额: $ 26.95万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-30 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6790613
• 关键词: animal tissue; cell adhesion; cell cell interaction; clinical research; corneal endothelium; endothelin; enzyme activity; fluid flow; focal adhesion kinase; gene expression; glaucoma; human tissue; immunofluorescence technique; intraocular aqueous flow; light microscopy; membrane permeability; myosin light chain kinase; organ culture; phosphorylation; polymerase chain reaction; postmortem; protein kinase; protein structure function; trabecular meshwork; transmission electron microscopy; western blottings

DESCRIPTION (provided by applicant): We hypothesize that Rho/Rho kinase regulated, actomyosin-mediated contraction and relaxation events in trabecular meshwork (TM), juxtacanalicular tissue (JCT) and Schlemm's canal (SC) cells modulate aqueous humor outflow resistance by changing the geometry of the flow pathway for aqueous humor, and/or by affecting paracellular fluid flow through the inner wall of Schlemm's canal. Strong support for this hypothesis derives from our recent studies, which demonstrate that augmentation and inhibition of Rho/Rho kinase function have contrasting effects not only upon cytoskeletal changes and myosin light chain (MLC) phosphorylation in human trabecular meshwork (HTM) and Schlemm's canal (HSC) cells, but on SC cell monolayer permeability characteristics and outflow facility as well. This convincing correlation between biochemical, morphological and functional attributes argues that Rho kinase dependent signaling pathway(s) represent a potential target for therapeutic modulation of outflow facility in glaucoma. To define the mechanistic basis(es) by which Rho/Rho kinase modulates aqueous outflow, we propose to study in detail, 1. The regulation of MLC phosphorylation in HTM, HSC and human ciliary muscle cells, and cytoskeletal organization (actin stress fibers, focal adhesions and adherens junctions) in HTM and HSC cells by using physiological (endothelin-1 and thromboxane A2, mimetic-U46619) and pharmacological (Y-27632 and HA-1077) modulators of Rho kinase activity and overexpression of a dominant negative mutant of Rho kinase (DNRK), 2. Modulation of paracellular permeability in HSC cell monolayers and of outflow facility in organ cultured post-mortem human eyes, using physiological agonists and pharmacological inhibitors of Rho kinase activity and overexpression of a DNRK. Changes in morphological integrity of outflow tissues will also be evaluated in perfused porcine and human eyes, 3. The ability of physiological factors and elevated ocular pressure to regulate Rho kinase expression in HTM and SC cells, and in perfused eyes, respectively, will be studied at the level of RNA and protein. The relevance of such changes for contractile function in TM will be also investigated. The completion of this work should provide significant insight into the role of the Rho/Rho kinase signaling pathway(s) in particular, and myosin phosphorylation and cellular contraction and relaxation in general, in regulating trabecular outflow facility. Identification of specific signaling mechanisms regulating function of outflow pathway tissues could enable the design of target-specific approaches to the treatment of glaucoma.

描述（由申请人提供）：我们假设小梁网（TM）、近小管组织（JCT）和施累姆氏管（SC）细胞中的Rho/Rho激酶调节、肌动球蛋白介导的收缩和松弛事件通过改变房水流动路径的几何形状和/或通过影响细胞旁液流动来调节房水流出阻力 穿过施莱姆运河的内壁。我们最近的研究有力地支持了这一假设，这些研究表明，Rho/Rho 激酶功能的增强和抑制不仅对人小梁网 (HTM) 和施累姆氏管 (HSC) 细胞中的细胞骨架变化和肌球蛋白轻链 (MLC) 磷酸化产生对比效应，而且对 SC 细胞单层通透性特征和流出设施也产生对比效应。生化、形态和功能属性之间令人信服的相关性表明，Rho 激酶依赖性信号通路代表了青光眼流出设施治疗调节的潜在靶标。为了明确 Rho/Rho 激酶调节房水流出的机制基础，我们建议详细研究，1. 通过使用生理学（内皮素-1 和 血栓素 A2，模拟-U46619）和 Rho 激酶活性的药理学（Y-27632 和 HA-1077）调节剂以及 Rho 激酶显性失活突变体 (DNRK) 的过度表达，2. 使用生理激动剂和 Rho 激酶活性和 DNRK 过度表达的药理学抑制剂。还将在灌注的猪和人眼中评估流出组织形态完整性的变化。 3. 将在 RNA 和蛋白质水平上研究生理因素和眼压升高分别调节 HTM 和 SC 细胞以及灌注眼中 Rho 激酶表达的能力。此类变化与 TM 收缩功能的相关性也将被研究。这项工作的完成将有助于深入了解 Rho/Rho 激酶信号通路在调节小梁流出设施中的作用，特别是肌球蛋白磷酸化和细胞收缩和舒张的作用。识别调节流出途径组织功能的特定信号机制可以帮助设计治疗青光眼的靶向特异性方法。