Determining How Lymphatic Molecules Control Conventional Outflow

项目总结/摘要 我们的目标是确定淋巴分子如何控制AQH引流和眼内压（IOP）升高。 眼压升高是青光眼的主要危险因素，青光眼是一种到2020年底将影响8000万人的疾病。 十年青光眼治疗是基于降低升高的IOP，但目前没有药物能够非常有效地降低IOP。 有效地表明迫切需要改进的疗法。IOP升高是由于对 房水（AQH）引流，发生在施累姆氏管内壁内皮附近 （SC）。然而，在SC内壁细胞中起作用的分子机制，以及产生抗性的分子机制， 到AQH引流，并没有很好的定义。小鼠和人类的SC在解剖学上非常相似， 因此，小鼠是研究SC功能的有力模型。使用 在小鼠中，我们发现SC是一种独特的血管，具有血液内皮和淋巴管内皮特征。 SC的一个关键特征是表达淋巴主调节转录因子Prox 1，这是需要的。 淋巴管的发育和维持。基于我们先前的研究表明，PROX 1富含于 SC内壁细胞，可能对这些细胞的功能特化很重要，并且对SC的已知功能也很重要。 淋巴组织中的PROX 1，Prox 1是我们功能研究的强有力候选者。在这个项目中，我们将 确定Prox 1在控制AQH引流中的作用。我们将通过三个目标来实现这一目标。目标1） 确定Prox 1单倍不足是否破坏AQH流出并升高IOP。在使用小鼠的初步研究中， 我们发现SC中的Prox 1异质性升高IOP。我们将在目标1中使用此工具来定义第一个 Prox 1在调节AQH引流和眼压中的作用。目的2）确定Prox 1敲除对细胞增殖的影响。 供应链的发展和功能。为了实现这一点，将在关键阶段的小鼠中有条件地删除Prox 1 SC的发展。这些实验将允许鉴定潜在的新的形态发生功能， Prox 1在发育中SC中的表达及成人SC中PROX 1功能的综合测定 确定SC中控制AQH流出或IOP的Prox 1调节通路。SC中的Prox 1调节基因 内壁是控制AQH流出的候选者。为了鉴定候选基因，我们将使用RNAseq和 差异表达分析以及靶向蛋白质组学，以确定Prox 1单倍性诱导的 导致IOP升高的SC变化。利用这些数据，候选途径和枢纽基因对 将识别AQH流出。重要的是，我们的项目将使用我们开发的创新方法， 使用Prox 1-GFP小鼠和其他荧光遗传标记对小鼠SC进行高分辨率检查， 报告子和小鼠中AQH流出的准确测量。成功实现这些目标将 产生关于调节IOP的机制的关键新信息，并将为识别 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.