Trabecular Meshwork Cytoskeletal Signaling-Regulation of Aqueous Humor Outflow

项目总结 导致房水流出阻力增加的体内平衡失调机制 通过小梁通路构成高眼压(OHT)的主要原因，这是 一种不可逆转的致盲疾病，青光眼。细胞外基质(ECM)被公认为起着至关重要的作用 通过调节眼球的收缩、粘附力和眼压，调节眼压和眼压 小梁流出道细胞的纤维化和通透性特征。仍然存在一种 然而，我们对ECM蛋白和ECM蛋白的翻译后修饰的理解存在根本差距 细胞外基质降解酶可能影响小梁网(TM)细胞行为、AH流出和眼压。这个 这项建议的主要目标是研究细胞外基质成分和基质的蛋白质磷酸化。 金属蛋白酶(MMPs)，并确定这种修饰如何调节AH流出和眼压，目的是 揭示OHT病因的新见解和开发治疗青光眼的有效策略。 科学前提是由TM和其他细胞分泌的AH中的蛋白酪氨酸激酶和磷酸酶 细胞类型通过修饰蛋白酪氨酸磷酸化在AH流出和IOP的动态平衡中起重要作用 ECM和MMPs的状态反过来影响TM细胞的收缩、黏附、纤维化和屏障活动。 我们假设，由于改变而导致的ECM蛋白和MMP酪氨酸磷酸化的解除管制 在分泌型酪氨酸激酶和磷酸酶的水平可能是一个关键的病因造成的损害 青光眼的眼压升高和流出。为了支持这一新颖的和范式转换的假设，我们最近的 研究检测到脊椎动物长链蛋白激酶(VLK)和磷酸酶和张力蛋白同源蛋白(PTEN)在 人的AH样本，证明HTM细胞分泌这两种酶。值得注意的是，VLK被证明是 调节细胞外基质酪氨酸磷酸化及转化生长因子-β-2诱导的TM细胞收缩和黏附活性 和地塞米松在人TM细胞中的作用，这两种物质都能诱导OHT。 基于这些有希望的和新颖的初步结果，我们勾勒出了这次竞赛的三个具体目标 建议，侧重于对VLK在以下方面的作用的机械性研究(S)：1)调节细胞内信号 TM细胞收缩、细胞-细胞外基质和细胞-细胞黏附、纤维化和通透性的调控途径 特性，2)TM细胞ECM和MMPs的Tyr-磷酸化，ECM组织和TM细胞硬度， (3)利用人TM细胞、人供体眼和大鼠的AH流出和眼压。这些研究将利用基因 靶向、蛋白质组学、生物物理学、组织学和生理学方法，并包括对 青光眼与非青光眼(白内障)患者AH标本中VLK和PTEN水平的变化 完成本申请书中建议的小说研究，可望揭示对 ECM蛋白和MMPs酪氨酸磷酸化在AH流出和由外向内改变的OHT病因中的作用 信号，并确定新的和有效的治疗策略，以降低青光眼患者的眼压。

PROJECT SUMMARY Dysregulated homeostatic mechanisms that lead to an increased resistance to aqueous humor (AH) outflow through the trabecular pathway constitute the main cause for ocular hypertension (OHT), a key risk factor for an irreversible blinding disease, glaucoma. The extracellular matrix (ECM) is well-recognized to play a crucial role in modulation of AH outflow and intraocular pressure (IOP) via regulating the contractile, adhesive, fibrogenic and permeability characteristics of the trabecular outflow pathway cells. There still exists a fundamental gap in our understanding however, of how post-translational modifications of ECM proteins and ECM degrading enzymes might influence trabecular meshwork (TM) cell behavior, AH outflow and IOP. The broad objective of this proposal is to examine protein phosphorylation of ECM components and matrix metalloproteinases (MMPs), and determine how this modification modulates AH outflow and IOP, with the goal of uncovering novel insights into OHT etiology and developing efficacious strategies for glaucoma treatment. The scientific premise is that protein tyrosine kinases and phosphatases in the AH secreted by TM and other cell types play a vital role in homeostasis of AH outflow and IOP by modifying protein tyr-phosphorylation status of ECM and MMPs that in turn influences TM cell contractile, adhesive, fibrogenic and barrier activities. We hypothesize that deregulation of tyr-phosphorylation of ECM proteins and MMPs resulting from alterations in the levels of secretory tyrosine kinases and phosphatases may be a key etiological contribution to impaired AH outflow and elevated IOP in glaucoma. In support of this novel and paradigm shifting hypothesis, our recent studies detected vertebrate lonesome kinase (VLK) and phosphatase and tensin homolog (PTEN) proteins in human AH samples, and demonstrated that HTM cells secrete both enzymes. Significantly, VLK was shown to regulate ECM tyr-phosphorylation, and contractile and adhesive activities of TM cells, and induced by TGF-β2 and dexamethasone in human TM cells, both of which are known to induce OHT. Based on these promising and novel preliminary results, we have outlined three specific aims in this competing proposal, focused on mechanistic investigations into the role(s) of VLK in: 1) regulation of intracellular signaling pathways controlling TM cell contractile, cell-ECM & cell-cell adhesive, fibrogenic and permeability characteristics, 2) Tyr-phosphorylation of TM cell ECM and MMPs, ECM organization and TM cell stiffness, and 3) AH outflow and IOP, using human TM cells, human donor eyes and rats. These studies will utilize gene targeting, proteomics, biophysical, histological and physiological approaches, and include an assessment of changes in levels of VLK and PTEN in AH samples from glaucoma versus non-glaucoma (cataract) patients. Completion of the novel studies proposed in this application is expected to uncover new understanding on the role of tyr-phosphorylation of ECM proteins and MMPs in AH outflow and OHT etiology via altered outside-in signaling, and to identify new and efficacious treatment strategies for lowering IOP in glaucoma patients.