The Role of Giant Vacuoles and Pores of Schlemm's Canal Endothelial Cells in Regulating Aqueous Outflow

摘要 原发性开角型青光眼是世界范围内致盲的主要原因。发展的主要风险因素 青光眼的进展是眼内压升高，这是由房水阻力增加引起的。 体液流出途径。大多数阻力被认为是在下小管结缔组织中产生的 组织（JCT）的血管内皮细胞，并受Schlemm管的内壁内皮细胞及其孔的调节。的机制 在正常眼和青光眼眼中调节房水流出阻力的机制尚不清楚， 新批准的药物针对这一耐药部位。穿过流出通道，水 体液通过施累姆氏管内皮细胞，形成短暂的、压力驱动的细胞外囊， 称为“巨泡”（GV）。GVs上的小孔允许房水进入施累姆氏 管从内皮。在青光眼中，先前的研究表明GV数量减少， 毛孔这个项目的总体目标是了解施累姆氏管的巨大空泡和孔隙的作用 内皮细胞调节房水流出。为此，创新技术将用于观察和 探索影响内皮细胞功能的可能因素（细胞骨架动力学和细胞连接）， 形成巨大空泡的能力。首先，一种新型的微流体装置将与 用活细胞成像来捕捉原发性Schlemm管内皮细胞的实时细胞骨架动力学， 3D细胞培养其次，将使用连续块面扫描电子显微镜分析数千个 沿着离体灌注的人供体眼睛的Schlemm管内壁的连续图像。与3D 重建细胞几何形状、内皮细胞形状及其与基础JCT的连接 细胞和基质将首次被分析和精确定量。细胞将在不同的 基于全局图像上的荧光示踪剂分布的流动类型（高、低和非流动区域）， 研究细胞连接如何影响流向周围某些区域的流量 的眼睛。本研究的具体目的是：1）观察巨泡形成过程中细胞骨架的动态变化 形成真实的时间，并确定单层渗透率和水力传导率响应于rho- 激酶抑制剂或地塞米松在原代Schlemm管内皮细胞中的作用; 和2）为了确定Schlemm管内皮细胞和底层细胞之间的细胞连接如何被激活， 大小管组织细胞/基质调节巨空泡和孔的形成及其对调控的贡献 节段性流出使用连续块面扫描电子显微镜。这些研究的结果将 推进我们对Schlemm管内皮细胞的巨大空泡和孔在 调节房水流出。这些新知识将有助于开发青光眼的新疗法， 靶向施累姆氏管的内壁内皮。

ABSTRACT Primary open-angle glaucoma is a leading cause of blindness worldwide. A primary risk factor for development and progression of glaucoma is elevation of intraocular pressure, caused by increased resistance in the aqueous humor outflow pathway. Most of the resistance is believed to be generated in the juxtacanalicular connective tissue (JCT) and modulated by the inner wall endothelium of Schlemm’s canal and its pores. The mechanisms that regulate aqueous outflow resistance remain unclear in normal and glaucomatous eyes, and only a couple newly-approved medications target this site of resistance. To traverse through the outflow pathway, aqueous humor passes through Schlemm’s canal endothelial cells in transient, pressure-driven cellular outpouchings, termed “giant vacuoles,” (GV). Pores, small openings in GVs, allow aqueous humor to enter into Schlemm’s canal from the endothelium. In glaucoma, previous studies have shown a reduction in the number of GV and pores. The overall goal of this project is to understand the role of giant vacuoles and pores of Schlemm’s canal endothelial cells in regulating aqueous outflow. To do this, innovative technology will be used to observe and explore possibile factors (cytoskeletal dynamics and cellular connectivity) that influence the endothelial cells’ ability to form giant vacuoles in new perspectives. First, a novel microfluidic device will be used in conjunction with live-cell imaging to capture real-time cytoskeletal dynamics of primary Schlemm’s canal endothelial cells in 3D cell culture. Secondly, serial block-face scanning electron microscopy will be used to analyze thousands of serial images along the inner wall of Schlemm’s canal of ex-vivo perfused human donor eyes. With 3D reconstruction of cellular geometries, the shapes of endothelial cells and their connections to underlying JCT cells and matrix will be analyzed and accurately quantified for the first time. Cells will be analyzed in different flow-types (high-, low-, and non-flow areas) based on fluorescent tracer distributions on global images to investigate how cellular connections affect the amount of flow going to certain regions around the circumference of the eye. The specific aims of this proposal are: 1) To observe cytoskeletal dynamics during giant vacuole formation in real time and determine monolayer permeability and hydraulic conductivity in response to a rho- kinase inhibitor or dexamethasone in primary Schlemm’s canal endothelial cells using a novel microfluidic device; and 2) To determine how cellular connections between Schlemm’s canal endothelial cells and underlying juxtacanalicular tissue cells/matrix modulate giant vacuole and pore formation and their contribution to regulation of segmental outflow using serial block-face scanning electron microscopy. The results of these studies will advance our understanding of the role of giant vacuoles and pores of Schlemm’s canal endothelial cells in regulating aqueous humor outflow. This new knowledge will help to develop new treatments for glaucoma by targeting the inner wall endothelium of Schlemm’s canal in the future.