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、肌动球蛋白介导的小梁网（TM）、关节旁组织（JCT）和施莱姆氏管（SC）细胞中的收缩和舒张事件通过改变房水流动通道的几何形状和/或通过影响通过施莱姆氏管内壁的细胞旁液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设，这些研究表明，Rho/Rho激酶功能的增强和抑制不仅对人类小梁网（HTM）和施莱姆管（HSC）细胞的细胞骨架变化和肌球蛋白轻链（MLC）磷酸化有不同的影响，而且对SC细胞单层通透性特征和流出设施也有不同的影响。这种生物化学、形态学和功能属性之间令人信服的相关性表明，Rho激酶依赖的信号通路代表了青光眼流出设施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。利用Rho激酶活性的生理（内皮素-1和血栓素A2、mimetics - u46619）和药理（Y-27632和HA-1077）调节剂和Rho激酶显性负突变体（DNRK）的过表达，调控HTM、HSC和人睫状肌细胞的MLC磷酸化，以及HTM和HSC细胞的细胞骨架组织（肌动蛋白应激纤维、局灶粘连和粘附连接），2。利用Rho激酶活性和DNRK过表达的生理激动剂和药理学抑制剂，调节死后人眼培养器官中HSC单层细胞旁通透性和流出设施。流出组织形态学完整性的变化也将在灌注的猪和人的眼睛中进行评估。生理因素和眼压升高对HTM细胞和SC细胞以及灌注眼中Rho激酶表达的调节能力将分别在RNA和蛋白水平上进行研究。这些变化与TM收缩功能的相关性也将被研究。这项工作的完成将对Rho/Rho激酶信号通路，以及肌球蛋白磷酸化和细胞收缩松弛在调节小梁流出设施中的作用提供重要的见解。确定调节流出通道组织功能的特定信号机制可以设计针对青光眼的靶向治疗方法。

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.