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导致失明的主要原因。发展的主要风险因素 青光眼的进展是由于房水中的阻力增加而引起的眼压升高。 幽默流出途径。大多数阻力被认为是在邻近的连接中产生的 组织(JCT)，并受Schlemm管及其毛孔的内壁内皮的调节。其作用机制 在正常和青光眼中，调节房水流出阻力的机制尚不清楚，只有几个 新批准的药物针对的是这一耐药部位。穿过流出通道，含水的 体液通过施莱姆管内皮细胞瞬间的、压力驱动的细胞突起， 称为“巨型空泡”(GV)。小孔，GV中的小开口，允许房水进入Schlemm 血管内皮细胞形成的管状结构。在青光眼中，以前的研究表明GV和GV的数量减少 毛孔。这个项目的总体目标是了解Schlemm管的巨大空泡和毛孔的作用 内皮细胞在房水流出调节中的作用。要做到这一点，将使用创新技术来观察和 探索影响内皮细胞的可能因素(细胞骨架动力学和细胞连通性) 在新的视角下形成巨大空泡的能力。首先，将结合使用一种新的微流控设备 应用活细胞成像技术实时捕捉原代Schlemm氏管内皮细胞的细胞骨架动力学 3D细胞培养。其次，将使用连续的块面扫描电子显微镜来分析数千个 沿着Schlemm管内壁的一系列图像显示了人体供体眼球。使用3D 重建细胞的几何形状、内皮细胞的形状及其与潜在的联合CT的连接 将首次对细胞和基质进行分析和准确量化。细胞将被分析在不同的 基于全球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特定区域的流量 眼睛的。该方案的具体目的是：1)观察巨型空泡中细胞骨架的动态变化 并确定响应Rho的单层渗透率和水力传导性。 使用一种新型微流控装置在原代Schlemm管内皮细胞中应用激酶抑制剂或地塞米松； 以及2)确定Schlemm管内皮细胞和基础细胞之间的细胞连接 颈旁组织细胞/基质对巨型空泡和孔洞形成的调控作用 用连续块面扫描电子显微镜观察节段性流出。这些研究的结果将 加深对Schlemm管内皮细胞巨大空泡和毛孔在血管内皮细胞损伤中作用的认识 调节房水流出。这一新知识将有助于通过以下方式开发青光眼的新疗法 未来将靶向Schlemm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.