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

• 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
• 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

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.

项目摘要 原发性开角型青光眼（POAG）是一种进行性视神经病变，可导致不可逆性失明。有 这种疾病有多种危险因素，但唯一可改变的属性是眼内压（IOP）升高。 IOP升高是由于前眼中的房水引流通路堵塞所致。诸如压力 建立，应力施加在后段的视神经乳头上，导致视神经损伤和 视力缓慢而不可逆转地下降。小梁网是负责调节眼压的组织 体内平衡作为对持续升高的压力的反应，TM细胞被拉伸，它们释放酶， 局部降解细胞外基质（ECM），从而允许更大的房水流出并降低IOP。青光眼 与年龄匹配的组织相比，TM具有过量的无序ECM，这表明功能障碍 ECM有助于增加对水流出的阻力。然而，目前尚不清楚哪种ECM 成分导致功能障碍。在本申请中，我们专注于一种称为血小板反应蛋白-1（TSP-1）的ECM蛋白。 1）。我们最近报道了一个单核苷酸多态性，rs 2228262，它导致错义N700 S TSP-1的氨基酸变化与一个POAG大家族的IOP升高显著相关。我们 初步数据显示，TSP-1 N700 S变异体存在于约9.5%的正常人和38%的 肉瘤TM细胞株。因此，我们有一个独特的机会来研究自然发生的 N700 S TSP-1变异体在肿瘤相关组织中的表达。我们假设N700 S TSP-1变体导致了 在ECM组织和TM细胞功能障碍中，这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起主要作用。然而，没有青光眼 针对调节TGFβ水平的治疗。在本申请中，我们将研究分子后果 的N700 S变异体在正常的和昏迷的TM细胞，并使用肽TSP-1拮抗剂靶向 TSP-1-TGFβ通路。我们的总体假设是TSP-1分子功能的改变破坏了ECM 正常和恶性肿瘤TM组织中TGFβ活性的变化。本文提出了三个目标：（1）分析 野生型和N700 S中的ECM组成、超微结构组织和TSP-1蛋白-蛋白相互作用 来源于正常和恶性肿瘤TM的变异细胞株;（2）测量N700 S变异体的影响 对吞噬作用、基质金属蛋白酶活性和TGFβ活性的影响， 表达和CRISPR编辑的TM细胞;和（3）使用肽拮抗剂，其竞争性和特异性地 通过TSP-1阻断TGFβ活化，以研究其对内源性和CRISPR编辑的N700 S TM细胞的影响， 以及确定其对IOP调节的离体模型中流出速率的影响。这些结果一起 将提供一个深入的分析，在TM中的一个与肺癌相关的TSP-1基因变异的作用，并将 探索以TSP-1-TGFβ通路为靶点的新疗法，以降低青光眼患者的高眼压。