Molecular Mechanisms of Outflow Segmentation and Intraocular Pressure Homeostasis

 描述（由申请人提供）：青光眼是失明的主要原因。眼内压（IOP）升高是青光眼视神经损伤的主要危险因素，降低IOP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IOP稳态的能力，是大多数青光眼的标志。因此，了解调节IOP稳态的机制（这是本提案的重点）对于改善这种常见致盲疾病的基于功能的治疗至关重要。 我们先前的研究指出了IOP调节的以下框架。由小梁网（TM）基质金属蛋白酶（MMPs）启动的细胞外基质（ECM）周转是维持房水流出阻力和IOP所必需的。响应于显著和持续的压力变化，流出路径启动IOP稳态响应，其中对流出阻力进行调节，从而将IOP恢复到窄的正常范围内。TM ECM周转是这一过程的核心。持续的压力变化被TM和/或施累姆氏管（SC）内壁的前小管区域内的细胞感知为机械拉伸或变形。然后，这些细胞启动ECM周转的复杂程序，以在几天的时间内调节流出阻力并将IOP恢复到可接受的范围内。此外，外流是高度节段性的周围的眼睛，这有戏剧性的后果，了解各方面的外流设施和IOP稳态的机制。 我们提出了两个目标，重点是阐明负责调节IOP稳态流出阻力调节的分子机制。这些研究将主要依赖于TM和SC细胞培养和灌注眼前节器官培养。具体目标1将需要在一系列时间点对1x与2x灌注压下的高流量区域与低流量区域进行详细的分子比较，在此期间使用正常眼和青光眼眼进行IOP稳态阻力调节。方法将包括：定量RT-PCR、PCR阵列、直接区域解剖和激光捕获显微解剖以及共聚焦免疫组织化学、Western免疫印迹和ELISA。具体目标2将确定调节区域流出阻力变化的信号转导途径，这些变化是响应于1x至2x压力变化而发生的。这将包括评估选择信号转导途径和转录调节剂的关键组分的活化状态。参与IOP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和基因过表达对途径组分进行遗传操作，以调节特定途径组分，从而影响IOP稳态过程。这种对正常眼和青光眼眼IOP稳态过程如何调节的详细分子和细胞理解将为恢复青光眼眼的这种稳态过程提供新的靶点。

 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.