Role of Rho Kinase in Aqueous Humor Outflow Pathway

描述（由申请人提供）：我们假设，在小梁网（TM）、小管组织（JCT）和施累姆氏管（SC）细胞中，Rho/Rho激酶调节的肌动球蛋白介导的收缩和舒张事件通过改变房水流动路径的几何形状和/或通过影响流经施累姆氏管内壁的细胞旁流体来调节房水流出阻力。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Schlemm管（HSC）细胞的细胞骨架变化和肌球蛋白轻链（MLC）磷酸化具有对比效应，而且对SC细胞单层渗透性特征和流出便利性也具有对比效应。生物化学、形态学和功能属性之间的这种令人信服的相关性表明，Rho激酶依赖性信号传导途径代表了青光眼流出功能治疗调节的潜在靶点。为了确定Rho/Rho激酶调节水流出的机制基础，我们建议详细研究，1。MLC磷酸化在HTM、HSC和人睫状肌细胞中的调节以及细胞骨架组织（肌动蛋白应力纤维，粘着斑和粘附连接）在HTM和HSC细胞中通过使用生理（内皮素-1和血栓素A2，模拟-U46619）和药理学（Y-27632和HA-1077）Rho激酶活性的调节剂和Rho激酶的显性失活突变体（DNRK）的过表达，2.使用Rho激酶活性的生理学激动剂和药理学抑制剂以及DNRK的过表达来调节HSC细胞单层中的细胞旁通透性和器官培养的死后人眼中的流出设施。还将在灌注的猪眼和人眼中评价流出组织形态完整性的变化，3。将在RNA和蛋白质水平研究生理因素和升高的眼压分别调节HTM和SC细胞以及灌注眼中Rho激酶表达的能力。还将研究这种变化与TM收缩功能的相关性。这项工作的完成将提供对Rho/Rho激酶信号通路的作用的重要见解，特别是肌球蛋白磷酸化和细胞收缩和松弛，在调节小梁流出设施。识别调节流出道组织功能的特定信号传导机制可以设计治疗青光眼的靶向特异性方法。

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.