Cellular Physiology of the Aqueous Outflow Pathway

描述(申请人提供)：青光眼是世界范围内导致失明的主要原因。原发性开角型青光眼(POAG)发生和发展的一个主要危险因素是由于房水流出阻力增加而引起的眼压升高。到目前为止，POAG中抵抗力升高的原因仍不清楚。以往的研究表明，房水流出阻力主要位于Schlemm管(SC)和管旁结缔组织(JCT)的内壁内皮附近。巨大的空泡和毛孔是SC内皮细胞的特征，长期以来一直被怀疑在调节流出阻力中发挥作用。POAG眼的巨大空泡和毛孔数量显著减少。JCT内细胞外基质(ECM)的异常积聚也被发现与POAG的阻力有关，但它们对总流出阻力的贡献尚不清楚。我们的长期目标是确定调节正常眼睛流出阻力的机制，以及POAG如何增加流出阻力。研究流出阻力的一个障碍是找到一个可以应用于各种物种的参数，而不考虑它们的流出解剖结构。我们的研究小组最近在小鼠、牛、猴和人的眼睛中发现，只有一小部分流出途径有效地促进了引流，并将这些流出区域称为有效滤过区(EFA)。我们已经证明，在牛、猴和人眼中，使用Rho-Kinase抑制剂后，EFA随着流出速度的增加而增加，而在急性和慢性眼压升高后，EFA则降低。此外，我们在低血压小鼠模型中发现了眼压和EFA之间的负相关关系。这些结果表明，EFA可作为不同物种的流出阻力和眼压的有价值的指标。我们开发了一种新的荧光团引导成像技术，以增加我们识别高和低血流区域之间重要的形态和细胞差异的机会，这些差异有助于增加流出阻力和POAG的发病机制。此外，我们现在能够使用一种新型的三维细胞培养设备实时观察SC内皮细胞的巨大空泡形成。通过这两种新的方法，我们将检验我们的假设，即SC内皮细胞与其潜在的ECM之间的相互作用调节巨型空泡和孔洞的形成，从而调节EFA和流出阻力。我们的具体目标也是：1)在高眼压动物模型中评估EFA与眼压之间的反向关系；2)确定正常人和POAG人眼的EFA与流出阻力之间的反向关系，并确定SC内皮细胞及其潜在的ECM在调节EFA中的作用；3)利用一种新型的实时成像三维细胞培养设备，确定SC内皮细胞及其潜在的ECM调节流出的机制。这项研究的结果将为一种新的治疗策略提供新的见解，以通过靶向小梁网络来降低眼压，小梁网络是最初的问题所在。

DESCRIPTION (provided by applicant): Glaucoma is a leading cause of blindness worldwide. A primary risk factor for the development and progression of primary open-angle glaucoma (POAG) is elevation of the intraocular pressure (IOP), resulting from a rise in aqueous humor outflow resistance. To date, the cause of this elevated resistance in POAG remains unknown. Previous studies have shown that the majority of aqueous outflow resistance is localized near the inner wall endothelium of Schlemm's canal (SC) and the juxtacanalicular connective tissue (JCT). Giant vacuoles and pores, which are characteristic features of the endothelial cells of SC, have long been suspected to play a role in regulating outflow resistance. A significant reduction in the number of giant vacuoles and pores was found in POAG eyes. Abnormal accumulations of extracellular matrix (ECM) within the JCT were also found contributing to resistance in POAG, but their contribution to total outflow resistance remains unknown. Our long-term goal is to determine the mechanisms that regulate outflow resistance in normal eyes and how this is increased in POAG. One obstacle in studying outflow resistance is finding a parameter that can be applied to various species regardless of their outflow anatomy. Our group has recently found, in mouse, bovine, monkey and human eyes, that only a fraction of outflow pathways actively contribute to drainage and termed those areas of outflow as the effective filtration area (EFA). We have demonstrated that EFA increases with higher outflow facility after use of a Rho-kinase inhibitor and decreases after acute and chronic elevation of IOP in bovine, monkey and human eyes. Moreover, we found an inverse relationship between IOP and EFA using a hypotensive mouse model. These results suggest that EFA serves as a valuable indicator across varied species for outflow resistance and IOP. We developed a novel fluorophore guided imaging technique to increase our chances of identifying important morphological and cellular differences between areas with high and low flow that contribute to increased outflow resistance and the pathogenesis of POAG. Additionally, we are now able to view giant vacuole formations of SC endothelial cells in real time using a novel three- dimensional cell culture device. With these two novel approaches, we will test our hypothesis that interactions between SC endothelial cells and their underlying ECM modulate giant vacuole and pore formations, thereby regulating EFA and outflow resistance. Our specific aims are too; 1) Evaluate the inverse relationship between the EFA and IOP in ocular hypertensive animal models; 2) Define the inverse relationship between the EFA and the outflow resistance in normal and POAG human eyes and determine the role of SC endothelial cells and their underlying ECM in regulating EFA; 3) Determine the mechanisms by which SC endothelial cells and their underlying ECM regulate the outflow using a novel three-dimensional cell culture device with real-time imaging. The results of this study will provide new insights for a novel therapeutic strategy to lower IOP by targeting the trabecular meshwork, where the initial problem resides.