understanding the mechanisms of pore formation and how pore formation may be induced with drugs may yield new insights into the pathogenesis and treat

青光眼是导致失明的主要原因之一，全世界约有6000万人受到影响(Quigley&Broman，2006年)，仅加勒比和拉丁美洲就有659万人(Tham等人，2014年；Allingham等人，1992年)。青光眼通常与眼压升高有关，目前所有青光眼治疗方法都致力于降低眼压。然而，许多治疗最终都失败了，导致失明的恶化，或者迫使患者接受高风险的青光眼引流手术。目前的青光眼药物可能会失败，因为它们不能有效地针对眼压升高的根本原因。尽管眼压调节的详细机制在很大程度上仍不清楚，但眼部对房水体液排出的水力阻力，即所谓的流出阻力，是眼压的主要决定因素。青光眼的流出阻力增加，导致眼压升高，这是青光眼的特征。流出阻力产生的来源位于Schlemm管(SC)内的细胞附近，SC是一种沿着虹膜外缘运行的血管，在从眼部排出时收集水体液(图1)。要进入SC，水体液必须穿过组成SC壁的连续细胞层。由于含水体液从细胞层下方向上流动，细胞会受到向上的力，将细胞推离其下面的支持组织，导致细胞形成被称为巨型空泡(GV)的穹顶状突起，通常与微米大小的毛孔有关(图1，右图)。这些孔为水的体液提供了穿过SC细胞壁的通道(Braakman 2015)。在青光眼中，毛孔的数量减少(Johnson等人，2002；Allingham等人，1992)，这可能解释了流出阻力增加的原因。因此，了解毛孔形成的机制以及药物如何诱导毛孔形成可能会为青光眼的发病机制和治疗提供新的见解。伦敦帝国理工学院的Overby实验室发现，毛孔形成是由机械力驱动的。当干细胞在GV形成期间被拉伸时，这种拉伸触发毛孔的形成(Braakman，2014)。在青光眼中，干细胞的生物力学硬度增加，并且这种增加的硬度与孔形成能力的降低相关(Overby等人，2014)。这表明，针对干细胞的生物力学特性，特别是细胞硬度的药物，可能对促进毛孔形成和治疗青光眼有用。然而，目前尚不清楚干细胞内的哪种特殊生物力学结构对孔形成的影响最大。该项目的目标是确定哪些亚细胞结构负责控制孔的形成。这些结构包括围绕细胞的细胞膜、细胞内称为细胞骨架的可收缩的丝状网络，以及细胞与其底层组织之间的粘连。这些结构中的每一种都可能影响生物力学硬度，从而影响SC细胞所经历的拉伸，从而影响孔的形成。我们假设，通过选择性地靶向SC细胞的特定生物力学成分，我们可能在药物上促进毛孔的形成。了解哪些细胞生物力学结构可以确定可以用于治疗青光眼的新药，例如使用FDA批准的药物来调节细胞膜的张力或收缩。目前，已经建立了一种在体外诱导SC细胞形成巨大空泡的设备(Pedrigi等人，2011年)，但目前还没有方法来测量SC细胞经历的拉伸。因此，这项拟议的工作将开发一种显微镜和图像分析系统，以定量测量干细胞在GV形成过程中经历的拉伸。我们将在SC细胞被有选择地改变T细胞的化合物处理时测量拉伸

Glaucoma is a leading cause of blindness that affects roughly 60 million people worldwide (Quigley & Broman, 2006), and 6.59 million people in the Caribbean and Latin America alone(Tham et al., 2014; Allingham et al., 1992). Glaucoma is typically associated with elevated eye pressure, which all current glaucoma treatments aim to reduce. However, many treatments eventually fail, allowing blindness to progress or forcing patients to undergo risky glaucoma drainage surgery. Current glaucoma medications likely fail because they do not effectively target the root cause of elevated eye pressure.Although the detailed mechanisms of eye pressure regulation remain largely unknown, the hydraulic resistance to aqueous humour drainage from the eye, known as outflow resistance, is the primary determinant of eye pressure. Outflow resistance is increased in glaucoma, leading to the elevated eye pressure that is characteristic of the disease. The source of outflow resistance generation lies near the cells lining Schlemm's Canal (SC), a vessel that runs along the outer border of the iris that collects aqueous humour as it drains from the eye (Figure 1).To enter SC, aqueous humour must cross the continuous cell layer that comprises SC wall. Because aqueous humour flows upwards from below the cell layer, the cells experience an upward-directed force that pushes the cells away from their underlying supporting tissue, causing the cells to form dome-shaped outpouchings known as giant vacuoles (GVs) that are typically associated with micrometer-sized pores (Figure 1, right panel). These pores provide the pathway for aqueous humour flow across the cellular wall of SC (Braakman 2015). In glaucoma, the number of pores is reduced (Johnson et al., 2002; Allingham et al., 1992), which may explain the source of increased outflow resistance. Thus, understanding the mechanisms of pore formation and how pore formation may be induced with drugs may yield new insights into the pathogenesis and treatment of glaucoma.The Overby lab at Imperial College London has discovered that pore formation is driven by mechanical force. As SC cells are stretched during GV formation, this stretch triggers pores to form (Braakman, 2014). In glaucoma, the biomechanical stiffness of SC cells increases, and the increased stiffness correlates with reduced pore-forming ability (Overby et al., 2014). This suggests that drugs that target the biomechanical properties of SC cells, particularly cell stiffness, may be useful to promote pore formation and treat glaucoma. However, it remains unclear which particular biomechanical structures within the SC cells would have the greatest effect on pore formation.The goal of this project is to determine which sub-cellular structures are responsible for controlling pore formation. These structures include the cell membrane surrounding the cell, the contractile filamentous network within the cell known as the cytoskeleton, and adhesions between the cell and its underlying tissue. Each of these structures may influence the biomechanical stiffness and hence the stretch experienced by SC cells to affect pore formation. We hypothesise that by selectively targeting specific biomechanical components of SC cells, we may pharmacologically promote pore formation. Knowing which cellular biomechanical structures to target will identify new drugs that could be used to treat glaucoma, for example using FDA-approved drugs that have already been shown to modulate cell membrane tension or contractility.Currently, an apparatus for inducing giant vacuole formation in SC cells in vitro has been established (Pedrigi et al., 2011), but there is currently no method to measure the stretch experienced by SC cells. The proposed work will thus develop a microscopy and image analysis system to quantitatively measure the stretch experienced by SC cells during GV formation. We will measure stretch as the SC cells are treated with compounds that selectively alter t