Molecular Mechanisms of Outflow Segmentation and Intraocular Pressure Homeostasis

 描述(申请人提供)：青光眼是导致失明的主要原因。眼压升高是青光眼视神经损伤的主要危险因素，降低眼压仍然是所有形式青光眼的唯一治疗方法。由于各种遗传或环境原因，丧失适当调节流出阻力的能力，即维持眼压动态平衡，是许多青光眼的特征。因此，了解调节眼压稳态的机制是改善这种常见致盲疾病的基于功能的治疗的核心，这是本提案的重点。我们之前的研究指出了以下眼压调节的框架。小梁网络(TM)基质金属蛋白酶(MMPs)启动的细胞外基质(ECM)转换是维持房水流出阻力和IOP的必要条件。当眼压发生显著且持续的变化时，流出通路会启动眼压稳态反应，对流出阻力进行调整，从而将眼压恢复到狭窄的正常范围内。TM ECM的周转是这一过程的核心。持续的压力变化由TM和/或Schlemm管(SC)内壁邻近区域的细胞感知为机械拉伸或变形。然后，这些细胞启动一个复杂的ECM周转程序，在几天的时间内调整流出阻力，并将眼压恢复到可接受的范围内。此外，眼球周围的流出是高度节段性的，这对理解流出设施的各个方面和眼压动态平衡的机制都有显著的影响。我们提出了两个目标，重点在于解开调节眼压稳态流出阻力调节的分子机制。这些研究将主要依靠TM和SC细胞培养以及眼前段器官灌流培养。具体目标1将需要对正常眼和青光眼在一系列时间点的1x和2x灌流压力下的高流量区和低流量区进行详细的分子比较，在这些时间点上，眼压平衡阻力正在发生调整。方法包括：定量RT-PCR、PCR阵列、直接区域剥离、激光捕获显微剥离以及共聚焦免疫组织化学、Western免疫印迹和ELISA。具体目标2将是确定调节区域流出阻力变化的信号转导通路，该变化响应于1x到2x的压力变化。这将包括评估选定的信号转导途径和转录调节因子的关键组件的激活状态。对参与眼压稳态过程的验证将包括使用途径抑制剂或激活剂，以及通过RNAi和基因过度表达对途径成分进行遗传操作，以调节特定的途径成分，从而影响眼压稳态过程。这种对正常和青光眼眼压平衡过程是如何调节的详细的分子和细胞理解将为恢复青光眼眼的这种平衡过程提供新的靶点。

 DESCRIPTION (provided by applicant): Glaucoma is a major cause of blindness. Elevated intraocular pressure (IOP) is the primary risk factor for glaucomatous optic nerve damage and reducing IOP remains the only treatment for all forms of glaucoma. Loss of the ability to appropriately regulate the outflow resistance, i.e. to maintain IOP homeostasis, due to a variety of genetic or environmental causes, is a hallmark of much of glaucoma. Understanding the mechanisms regulating IOP homeostasis, which is the focus of this proposal, is thus central to improved function based therapy for this common blinding disease. Our prior studies point to the following framework for IOP regulation. Extracellular matrix (ECM) turnover, initiated by trabecular meshwork (TM) matrix metalloproteinases (MMPs) is required to maintain the aqueous humor outflow resistance and thus IOP. In response to a significant and sustained pressure change, the outflow pathway initiates an IOP homeostatic response in which adjustments are made to the outflow resistance, thus restoring IOP to within a narrow normal range. TM ECM turnover is central to this process. Sustained pressure changes are sensed by cells within the juxtacanalicular region of the TM and/or Schlemm's canal (SC) inner wall as mechanical stretching or distortion. These cells then initiated a complex program of ECM turnover to adjust the outflow resistance over several days' time and restore IOP to within acceptable bounds. In addition, outflow is highly segmental around the circumference of the eye, which has dramatic consequences for understanding all aspects of outflow facility and the mechanisms of IOP homeostasis. We propose two aims focused on unraveling the molecular mechanisms responsible for regulating IOP homeostatic outflow resistance adjustments. These studies will rely primarily on TM and SC cell culture and perfused anterior segment organ culture. Specific Aim 1 will entail detailed molecular comparisons of high flow regions with low flow regions for 1x vs. 2x perfusion pressures at a series of time points during which the IOP homeostatic resistance adjustment is occurring using both normal and glaucoma eyes. Methods will include: quantitative RT-PCR, PCR arrays, direct regional dissection, and laser capture microdissection as well as confocal immunohistochemistry, Western immunoblots, and ELISAs. Specific Aim 2 will be to identify the signal transduction pathways that regulate the regional outflow resistance changes that occur in response to the 1x to 2x pressure change. This will include assessing activation states of key components of select signal transduction pathways and transcriptional modulators. Verification of involvement in the IOP homeostatic process will include using pathway inhibitors or activators and genetic manipulation of pathway components via RNAi and gene overexpression to modulate specific pathway components and thus affect the IOP homeostatic process. This detailed molecular and cellular understanding of how the IOP homeostatic process is regulated in normal and glaucomatous eyes will provide new targets to restore this homeostatic process in glaucomatous eyes.