Thrombospondin-1 in normal and glaucomatous trabecular meshwork

正常和青光眼小梁网中的血小板反应蛋白-1

基本信息

批准号：
10444384
负责人：
Kate E Keller
金额：
$ 40.93万
依托单位：
OREGON HEALTH & SCIENCE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-07-01 至 2026-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10444384
关键词：
Affect Age Amino Acids Anterior Aqueous Humor Binding Binding Proteins Biological Assay Biological Markers Blindness Calcium Binding Cell physiology Cells Characteristics Chronic Clustered Regularly Interspaced Short Palindromic Repeats Complex Confocal Microscopy Data Development Disease Drainage procedure Electron Microscopy Enzymes Extracellular Matrix Extracellular Matrix Proteins Eye Family Functional disorder Gelatinase A Genetic Genotype Glaucoma Heparin Binding Homeostasis Human In Situ In Vitro Integrin Binding Integrin alphaVbeta3 Knockout Mice Ligation Matrix Metalloproteinases Measures Mechanics Modeling Modification Molecular Optic Disk Optic Nerve Pathway interactions Patients Peptides Persons Phagocytosis Physiologic Intraocular Pressure Play Primary Open Angle Glaucoma Proteins Publications Recombinant Proteins Regulation Reporting Resistance Reverse Transcriptase Polymerase Chain Reaction Risk Factors Role Single Nucleotide Polymorphism Stress Stretching THBS1 gene Testing Therapeutic Thrombospondin 1 Tissues Trabecular meshwork structure Transforming Growth Factor Beta 2 Transforming Growth Factor beta Transforming Growth Factors United States Variant Vision Western Blotting antagonist aqueous base cell type design effective therapy enzyme activity genetic variant insight mRNA Expression nerve damage new therapeutic target normotensive novel optic nerve disorder overexpression pressure prevent protein protein interaction response

项目摘要

Project Summary Primary open-angle glaucoma (POAG), a progressive optic neuropathy, causes irreversible blindness. There are multiple risk factors for this disease, but the only modifiable attribute is elevated intraocular pressure (IOP). Elevated IOP results from a blockage in the aqueous humor drainage pathway in the anterior eye. As pressure builds, stress is exerted on the optic nerve head in the posterior segment, leading to optic nerve damage and a slow and irreversible decline in vision. The trabecular meshwork (TM) is the tissue responsible for regulating IOP homeostasis. In response to sustained elevated pressure, TM cells are stretched and they release enzymes to focally degrade extracellular matrix (ECM), which allows greater aqueous outflow and lowers IOP. Glaucomatous TM has excess amounts of disorganized ECM compared to age-matched tissue, suggesting that dysfunctional ECM contributes to increased resistance to aqueous outflow. However, it is currently unknown which ECM components cause dysfunction. In this application, we focus on one ECM protein called thrombospondin-1 (TSP- 1). We recently reported that a single nucleotide polymorphism, rs2228262, which causes a missense N700S amino acid change in TSP-1, was significantly associated with elevated IOP in a large POAG family. Our preliminary data show that the TSP-1 N700S variant is present in approximately 9.5% of normal and 38% glaucomatous TM cell strains. Thus, we have a unique opportunity to study the function of the naturally occurring N700S TSP-1 variant in a glaucoma-relevant tissue. We postulate that the N700S TSP-1 variant causes changes in ECM organization and TM cell dysfunction, which contribute to development of fibrotic matrices characteristic of the glaucomatous TM in situ. TSP-1 is a potent activator of transforming growth factor-β (TGFβ), which stimulates ECM synthesis and plays major roles in glaucoma pathophysiology. Yet, there are no glaucoma therapies directed at modulating TGFβ levels. In this application, we will investigate the molecular consequences of the N700S variant in normal and glaucomatous TM cells, and use a peptide TSP-1 antagonist to target the TSP-1-TGFβ pathway. Our overall hypothesis is that modifications to TSP-1 molecular function disrupt ECM organization and TGFβ activity in the normal and glaucomatous TM. Three aims are proposed: (1) to analyze ECM composition, ultrastructural organization and TSP-1 protein-protein interactions in wild-type and N700S variant cell strains derived from normal and glaucomatous TM; (2) to measure the impact of the N700S variant on phagocytosis, matrix metalloproteinase enzyme activity, and TGFβ activity using endogenous, over- expressed and CRISPR-edited TM cells; and (3) to use a peptide antagonist, which competitively and specifically blocks TGFβ activation by TSP-1, to investigate its effects on endogenous and CRISPR-edited N700S TM cells, as well as to determine its effects on outflow rates in an ex vivo model of IOP regulation. Together, these results will provide an in-depth analysis of the role of a glaucoma-associated TSP-1 gene variant in the TM and will explore a novel therapeutic targeting the TSP-1-TGFβ pathway to reduce elevated IOP in glaucoma patients.

项目概要原发性开角型青光眼（POAG）是一种进行性视神经病变，会导致不可逆的失明。这种疾病有多种危险因素，但唯一可改变的因素是眼内压（IOP）升高。眼压升高是由于前眼房水引流通路阻塞所致。形成后，对后段的视神经乳头施加压力，导致视神经损伤和视力缓慢且不可逆转的下降小梁网 (TM) 是负责调节 IOP 的组织。为了应对持续升高的压力，TM 细胞会被拉伸并释放酶以维持体内平衡。局部降解细胞外基质 (ECM)，从而增加房水流出并降低眼压。与年龄匹配的组织相比，TM 具有过量的杂乱的 ECM，这表明功能失调的 ECM 有助于增加水流出的阻力，但目前尚不清楚是哪种 ECM。在此应用中，我们重点关注一种称为血小板反应蛋白-1 (TSP-) 的 ECM 蛋白。 1）我们最近报道了一个单核苷酸多态性，rs2228262，它导致了N700S的错义。在我们的 POAG 大家族中，TSP-1 的氨基酸变化与眼压升高显着相关。初步数据显示，TSP-1 N700S 变体存在于大约 9.5% 的正常细胞和 38% 的正常细胞中。因此，我们有一个独特的机会来研究天然存在的 TM 细胞株的功能。青光眼相关组织中的 N700S TSP-1 变异我们假设 N700S TSP-1 变异导致变化。 ECM 组织和 TM 细胞功能障碍，有助于纤维化基质特征的发展青光眼 TM 的原位 TSP-1 是转化生长因子-β (TGFβ) 的有效激活剂。刺激 ECM 合成并在青光眼病理生理学中发挥重要作用然而，青光眼并不存在。旨在调节 TGFβ 水平的疗法在本申请中，我们将研究分子后果。正常和青光眼 TM 细胞中的 N700S 变体，并使用肽 TSP-1 拮抗剂来靶向我们的总体假设是 TSP-1 分子功能的改变会破坏 ECM。提出了三个目标：（1）分析正常和青光眼 TM 中的组织和 TGFβ 活性。野生型和 N700S 中的 ECM 组成、超微结构组织和 TSP-1 蛋白质-蛋白质相互作用 (2) 测量 N700S 变体的影响使用内源性、过度的吞噬作用、基质金属蛋白酶活性和 TGFβ 活性表达和 CRISPR 编辑的 TM 细胞；以及 (3) 使用肽拮抗剂，该肽拮抗剂具有竞争性和特异性通过 TSP-1 阻断 TGFβ 激活，以研究其对内源性和 CRISPR 编辑的 N700S TM 细胞的影响，以及在 IOP 调节的离体模型中确定其对流出率的影响。将深入分析青光眼相关 TSP-1 基因变异在 TM 中的作用，并将探索一种针对 TSP-1-TGFβ 途径的新型治疗方法，以降低青光眼患者升高的眼压。