Molecular Mechanisms of Outflow Segmentation and Intraocular Pressure Homeostasis

流出分段和眼压稳态的分子机制

• 批准号: 9115612
• 负责人: TED S ACOTT
• 金额: $ 34.65万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-01 至 2019-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9115612
• 关键词: Affect; Anterior; Aqueous Humor; Blindness; Cell Culture Techniques; Cells; Complex; Disease; Dissection; Elderly; Enzyme-Linked Immunosorbent Assay; Extracellular Matrix; Eye; Family suidae; Gene Expression; Genes; Genetic; Glaucoma; Health; Homeostasis; Hour; Human; Immunohistochemistry; Knowledge; Label; Left; Link; Matrix Metalloproteinases; Measures; Mechanics; Methods; Molecular; Molecular Analysis; Molecular Profiling; Normal Range; Optic Nerve; Organ Culture Techniques; Pathway interactions; Pattern; Perfusion; Persons; Physiologic Intraocular Pressure; Process; Proteins; RNA Interference; Regulation; Research; Resistance; Reverse Transcriptase Polymerase Chain Reaction; Risk Factors; Series; Signal Transduction Pathway; Stretching; Structure of sinus venosus of sclera; Testing; Therapeutic; Time; Trabecular meshwork structure; Tracer; Transcript; Western Blotting; Work; base; genetic manipulation; improved functioning; inhibitor/antagonist; laser capture microdissection; novel; overexpression; pressure; programs; research study; response; restoration

 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.

 描述（由申请人提供）：青光眼是导致失明的主要原因。眼内压升高是青光眼视神经损伤的主要危险因素，降低眼内压仍然是治疗所有形式青光眼的唯一方法。由于各种遗传或环境原因，调节流出阻力，即维持眼压稳态，是了解青光眼机制的一个标志。因此，调节 IOP 稳态是本提案的重点，对于改善这种常见致盲疾病的基于功能的治疗至关重要，我们之前的研究指出了以下由小梁网启动的 IOP 调节框架。 TM）基质金属蛋白酶（MMP）需要维持房水流出阻力，从而维持眼压。响应于显着且持续的压力变化，流出通路启动眼压稳态反应，从而进行调整。 TM 和/或施莱姆管 (SC) 内壁的近管区细胞可感知持续的压力变化。然后，这些细胞启动了复杂的 ECM 更新程序，以在几天的时间内调整流出阻力，并将 IOP 恢复到可接受的范围内。此外，流出在周围是高度分段的。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。 SC 细胞培养和灌注前段器官培养需要在 IOP 期间的一系列时间点对 1x 与 2x 灌注压力的高流量区域与低流量区域进行详细的分子比较。使用正常和青光眼眼睛进行稳态阻力调整，方法包括：定量 RT-PCR、PCR 阵列、直接区域解剖、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。确定调节响应 1 至 2 倍压力变化而发生的区域流出阻力变化的信号转导途径，这将包括评估所选信号关键成分的激活状态。转导途径和转录调节剂参与 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成分进行遗传操作，以调节特定途径成分，从而影响 IOP 稳态过程。了解正常眼和青光眼眼中眼压稳态过程的调节机制将为恢复青光眼眼中的这种稳态过程提供新的目标。