The Mechanical Basis of Primary Open Angle Glaucoma

 描述（由申请人提供）：我们提出原发性开角型青光眼的眼内压（IOP）升高是由于施累姆氏管（SC）内皮细胞硬度增加所致，这损害了孔形成，从而阻碍了房水流出。这一假说是机制性的，可检验的，并且当被利用时可以导致青光眼的新疗法。在本BRP的第一个授予期内获得的数据实现了所有设定的里程碑，并有力地支持了中心假设。我们已经表明，SC细胞是高度收缩性的，修改其收缩应力和刚度的水平与平滑肌细胞相当。我们已经证明，SC细胞上的机械应变增强孔形成。我们还发现了细胞硬度和流出阻力之间的显着联系，特别是增加（减少）SC细胞硬度的药物增加（减少）阻力。总之，这些观察结果表明，SC细胞是高度机械敏感的，它们的生物力学活性与水流出调节和在机械要求高的环境中的存活密切相关。此外，我们的研究进一步证明，肿瘤SC细胞具有改变的机械生物学，包括：（i）升高的细胞刚度，（ii）降低的成孔能力，和（iii）增强的对基质刚度的机械敏感性。我们的后一个发现与其他人最近的发现特别相关，后者显示青光眼眼小梁网硬度升高。我们的更新申请建立在这些里程碑的基础上，重点关注机制，遗传学和治疗应用的翻译。为了验证我们的假设，我们设计了五个具体目标：首先，我们将扩展我们已经开发的概念模型，该模型详细描述了细胞刚度和流出阻力之间的关系。其次，我们将把我们在体外发现的昏迷SC细胞硬度升高的开创性发现扩展到原位情况， 并考察其在生理负荷下是否被放大。第三，我们将确定基因调控过程在昏迷SC细胞生物力学改变中的作用，探索潜在的机制。第四，我们将引入高通量功能（机械生物学为基础）筛选，以确定新的候选化合物，减少SC细胞收缩力。最后，我们将使用病毒载体来传递细胞硬度改变基因，该基因特异性靶向SC（而不是TM或其他细胞类型），并监测对流出功能的影响。我们的假设的测试将使合理的发展有针对性的青光眼治疗，选择性地降低细胞硬度在施累姆氏管的水平，从而降低IOP。

 DESCRIPTION (provided by applicant): We propose that intraocular pressure (IOP) elevation in primary open angle glaucoma results from increased stiffness of Schlemm's canal (SC) endothelial cells, which impairs pore formation, and consequently obstructs aqueous humor outflow. This hypothesis is mechanistic, testable and when exploited can lead to novel therapies for glaucoma. Data obtained during the first award period of this BRP achieved all set milestones, and strongly supports the central hypothesis. We have shown that SC cells are highly contractile, modifying their contractile stresses and stiffnesses to levels comparable to smooth muscle cells. We have demonstrated that mechanical strain on SC cells potentiates pore formation. We have also discovered a remarkable link between cell stiffness and outflow resistance, specifically that drugs that increase (decrease) SC cell stiffness increase (decrease) resistance. Together, these observations demonstrate that SC cells are highly mechanosensitive and their biomechanical activity is tightly tied to aqueous outflow regulation and survival in a mechanically demanding environment. Moreover, our studies have further demonstrated that glaucomatous SC cells have altered mechanobiology including: (i) elevated cell stiffness, (ii) reduced pore-forming capability, and (iii) enhanced mechanosensitivity to substrate stiffness. This latter finding of ours is particularly relevant to recent findings of othrs, showing elevated stiffness in the trabecular meshwork of glaucomatous eyes. Our renewal application builds upon these milestones and focuses upon mechanism, genetics and translation to therapeutic applications. To test our hypothesis, we have designed five specific aims: First, we will extend a conceptual model we have developed that details the relationship between cell stiffness and outflow resistance. Second, we will look to extend our seminal findings of elevated stiffness of glaucomatous SC cells in vitro to the situation in situ, and will also examine whether the effect is magnified under physiological load. Third, we will determine the role of genetic regulatory processes in the altered biomechanics of glaucomatous SC cells, exploring underlying mechanisms. Fourth, we will introduce high-throughput functional (mechanobiologically based) screening to identify new candidate compounds that decrease SC cell contractile forces. Finally, we will use viral vectors to deliver cell stiffness-altering gene that specifically target the SC (and not TM, or other cell types) and monitor effects on outflow function. Testing of our hypothesis will enable rational development of targeted glaucoma therapies that selectively decrease cell stiffness at the level of Schlemm's canal, consequently reducing IOP.