Role of Rho Kinase in Aqueous Humor Outflow Pathway

描述(由申请人提供)：我们假设Rho/Rho激酶调节小梁网(TM)、小梁旁组织(JCT)和Schlemm管(SC)细胞的肌动蛋白介导的收缩和松弛事件，通过改变房水的流动路径的几何形状和/或通过影响Schlemm管内壁的细胞旁液体流动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说，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络(HTM)和Schlemm管(HSC)细胞的细胞骨架变化和肌球蛋白轻链(MLC)磷酸化有相反的影响，而且对SC细胞的单层通透性和流出能力也有相反的影响。生化、形态和功能属性之间的这种令人信服的相关性表明，Rho激酶依赖的信号通路(S)代表了青光眼流出设施治疗调节的潜在靶点。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.生理(内皮素-1和血栓素A2，Mimtic-U46619)和药物(Y-27632和HA-1077)调节Rho激酶活性和优势负性突变体(DNRK)的过度表达；2.生理(ET-1和血栓素A2，模拟U46619)和药物(Y-27632和HA-1077)对HSC细胞单层细胞通透性和细胞外流设施的调节；使用生理激动剂和药物抑制剂来抑制Rho激酶活性和DNRK的过度表达。3.生理因素和高眼压分别在RNA和蛋白质水平上研究生理因素和高眼压对HTM和SC细胞以及灌注眼的Rho激酶表达的调节能力。这些变化与TM收缩功能的相关性也将被调查。这项工作的完成将为深入了解Rho/Rho激酶信号通路(特别是S)以及肌球蛋白磷酸化和细胞收缩和松弛在调节小梁流出设施中的作用提供重要的见解。识别调节流出道组织功能的特定信号机制可以使设计靶向特异性的青光眼治疗方法成为可能。

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.