Determining How Lymphatic Molecules Control Conventional Outflow

项目摘要/摘要 我们的目标是确定淋巴分子如何控制AQH引流和眼压升高。 眼压升高是青光眼的主要危险因素，到2019年年底，青光眼将影响8000万人。 十年。青光眼治疗的基础是降低高眼压，但目前还没有一种药物能够非常有效地降低眼压 有效地表明了改进治疗的迫切需要。眼压升高是由于对 房水引流，发生在Schlemm管内壁内皮附近 (SC)。然而，在SC内壁细胞中起作用的分子机制，以及在产生抗性 到AQH排水，都没有很好的定义。小鼠和人类的SC在解剖学上非常相似，而且 AQH流出生理学方面，小鼠是研究SC功能的有力模型。使用 小鼠，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的特征。 SC的一个关键特征是表达淋巴管主控转录因子Prox1，这是必需的 用于淋巴发育和维护。基于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和已知的功能很重要。 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引流中的作用。我们将通过三个目标实现这一目标。目标1) 确定Prox1单倍体功能不全是否会扰乱AQH的流出并升高眼压。在用小鼠进行的初步研究中 我们发现在SC中Prox1的异质性会升高眼压。我们将在目标1中使用此工具为第一个 Prox1在调节AQH引流和眼压中的作用。目的2)确定Prox1基因敲除对 供应链的发展和作用。为了做到这一点，Prox1将在小鼠的关键阶段有条件地被删除 合作伙伴关系的发展。这些实验将允许识别潜在的新的形态发生函数 PROX1在SC发育中的作用及成人SC中PROX1功能的综合测定目标3) 确定在SC中Prox1调节的控制AQH外流或IOP的通路。干细胞中受PROX1调控的基因 内壁是控制AQH流出的候选对象。为了确定候选基因，我们将使用RNAseq和 差异表达分析和靶向蛋白质组学确定Prox1单倍体功能不全 引起眼压升高的SC的变化。使用这些数据，候选路径和枢纽基因对 将确定AQH流出情况。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进行高分辨率检测 并准确测量AQH在小鼠体内的流出情况。这些目标的成功实现将 获得有关眼压调节机制的关键新信息，并将为识别奠定基础 更有效的青光眼治疗靶点。

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.