Determining How Lymphatic Molecules Control Conventional Outflow

确定淋巴分子如何控制常规流出

• 批准号: 9564676
• 负责人: Krishnakumar Kizhatil
• 金额: $ 43.75万
• 依托单位: JACKSON LABORATORY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-30 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9564676
• 关键词: Adult; Affect; Anatomy; Antibodies; Aqueous Humor; Biological; Biology; Blindness; Blood; Blood Vessels; Candidate Disease Gene; Cell Wall; Cells; Confocal Microscopy; Data; Defect; Development; Devices; Dimensions; Disease; Drainage procedure; Endothelium; Eye; Gene Deletion; Gene Expression; Genes; Genetic; Glaucoma; Goals; Heterozygote; Human; Imagery; Knock-out; Knowledge; Lymphatic; Lymphoid Tissue; Maintenance; Measurement; Measures; Mediating; Methods; Modeling; Molecular; Morphology; Mouse Strains; Mus; Ocular Hypertension; Pathway interactions; Patients; Perfusion; Pharmaceutical Preparations; Phenotype; Phosphorylation; Physiologic Intraocular Pressure; Physiological; Physiology; Positioning Attribute; Process; Proteins; Proteomics; Public Health; Regulation; Reporter; Resistance; Resolution; Risk Factors; Role; Route; Signal Pathway; Structure of sinus venosus of sclera; Study models; System; Tamoxifen; Techniques; Testing; Time; Tracer; Treatment Efficacy; Vision; anterior chamber; aqueous humor flow; base; differential expression; effective therapy; experimental study; fluid flow; genome-wide; high resolution imaging; improved; innovation; new therapeutic target; novel; novel strategies; response; therapeutic target; tool; transcription factor; transcriptome sequencing

PROJECT SUMMARY/ABSTRACT Our goal is to define how lymphatic molecules control AQH drainage and intraocular pressure (IOP) elevation. IOP elevation is a major risk factor for glaucoma, a disease that will affect 80 million people by the end of the decade. Glaucoma therapy is based on reducing elevated IOP but no current drug is able to reduce IOP very effectively indicating the pressing need for improved therapies. IOP elevation results from resistance to aqueous humor (AQH) drainage, which occurs in the vicinity of the inner wall endothelium of Schlemm's canal (SC). However, the molecular mechanisms functioning in the SC inner wall cells, and in generating resistance to AQH drainage, are not well defined. The SC of mice and humans are very similar anatomically and with respect to AQH outflow physiology; thus the mouse is a powerful model for studying SC function. Using the mouse, we showed that SC is a unique vessel with both blood endothelial and lymphatic endothelial features. A key feature of SC is expression of the lymphatic master regulator transcription factor Prox1, which is required for lymphatic development and maintenance. Based on our previous study showing that PROX1 is enriched in SC inner wall cells and is likely important for functional specialization of these cells, and on known functions of PROX1 in lymphatic tissues, Prox1 is a strong candidate for our functional studies. In this project we will determine the role of Prox1 in controlling AQH drainage. We will accomplish this in three aims. Aim 1) To determine if Prox1 haploinsufficiency disrupts AQH outflow and raises IOP. In preliminary studies using mice we show that Prox1 heterozgosity in the SC elevates IOP. We will use this tool in Aim 1 to define for the first time the role of Prox1 in regulating AQH drainage and IOP. Aim 2) Determine the effect of Prox1 knockout on SC development and function. To accomplish this, Prox1 will be deleted conditionally in mice at critical stages of SC development. These experiments will allow identification of potentially new morphogenetic functions for Prox1 in the developing SC and comprehensive determination of PROX1 function in the adult SC. Aim 3) Identify Prox1-regulated pathways in SC that control AQH outflow or IOP. Prox1-regulated genes in the SC inner wall are candidates for controlling AQH outflow. To identify candidate genes we will use RNAseq and differential expression analysis as well as targeted proteomics to define Prox1 haploinsufficiency-induced changes in SC that cause IOP elevation. Using these data, candidate pathways and hub genes important for AQH outflow will be identified. Importantly, our project will use innovative new approaches that we developed for high-resolution examination of the mouse SC using the Prox1-GFP mouse and other fluorescent genetic reporters and accurate measurement of AQH outflow in the mouse. Successful completion of these aims will yield critical new information on the mechanisms regulating IOP, and will lay the groundwork for identification of more effective therapeutic targets for glaucoma.

项目概要/摘要 我们的目标是确定淋巴分子如何控制 AQH 引流和眼压 (IOP) 升高。 眼压升高是青光眼的主要危险因素，到本世纪末，这种疾病将影响 8000 万人。 十年。青光眼治疗的基础是降低升高的眼压，但目前没有药物能够显着降低眼压 有效地表明了改进治疗方法的迫切需要。眼压升高是由于抵抗 房水 (AQH) 引流，发生在施累姆氏管内壁内皮附近 （SC）。然而，SC 内壁细胞中发挥作用以及产生抵抗力的分子机制 AQH 排水，没有明确定义。小鼠和人类的 SC 在解剖学上非常相似，并且 尊重 AQH 流出生理学；因此，小鼠是研究 SC 功能的强大模型。使用 在小鼠实验中，我们发现 SC 是一种独特的血管，同时具有血液内皮和淋巴管内皮特征。 SC 的一个关键特征是淋巴主调节转录因子 Prox1 的表达，这是必需的 用于淋巴管的发育和维持。根据我们之前的研究表明 PROX1 富含 SC 内壁细胞，可能对这些细胞的功能特化以及已知的功能很重要 PROX1 在淋巴组织中，Prox1 是我们功能研究的有力候选者。在这个项目中我们将 确定 Prox1 在控制 AQH 排水中的作用。我们将通过三个目标来实现这一目标。目标 1) 至 确定 Prox1 单倍体不足是否会扰乱 AQH 流出并提高 IOP。在使用小鼠的初步研究中 我们发现 SC 中的 Prox1 异质性会升高 IOP。我们将在目标 1 中使用此工具来定义第一个 Prox1 在调节 AQH 引流和 IOP 中的作用。目标 2) 确定 Prox1 敲除对 SC的发展和功能。为了实现这一目标，Prox1将在关键阶段的小鼠体内有条件地被删除 SC 开发。这些实验将能够识别潜在的新形态发生功能 发育中 SC 中的 Prox1 以及成人 SC 中 PROX1 功能的综合测定。目标 3) 确定 SC 中控制 AQH 流出或 IOP 的 Prox1 调节途径。 SC 中 Prox1 调控的基因 内壁是控制 AQH 流出的候选者。为了识别候选基因，我们将使用 RNAseq 和 差异表达分析以及靶向蛋白质组学来定义 Prox1 单倍体不足诱导的 SC 的变化导致 IOP 升高。使用这些数据、候选途径和中心基因对于 AQH 流出将被识别。重要的是，我们的项目将使用我们开发的创新方法 使用 Prox1-GFP 小鼠和其他荧光基因对小鼠 SC 进行高分辨率检查 记者并准确测量小鼠 AQH 流出量。成功完成这些目标将 产生有关 IOP 调节机制的重要新信息，并将为识别奠定基础 寻找更有效的青光眼治疗靶点。