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（适用提供）：青光眼是失明的主要原因。眼内压（IOP）是青光眼视神经损伤的主要风险因素，而减少IOP仍然是所有形式的青光眼的唯一治疗方法。丧失适当调节输出流抗性的能力，即由于多种遗传或环境原因，维持IOP稳态的能力是大部分青光眼的标志。因此，了解调节IOP体内稳态的机制，这是该提案的重点，因此对于这种常见的盲目疾病的基于功能的治疗是至关重要的。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（MMP）引发的细胞外基质（ECM）周转率是为了维持水性幽默出口电阻，因此需要IOP。为了响应重大和持续的压力变化，出口途径启动了IOP稳态反应，在该响应中，对出口电阻进行了调整，从而将IOP恢复到狭窄的正常范围内。 TM ECM营业额是此过程的核心。在TM的近距离区域内和/或Schlemm的内壁（SC）内壁中的细胞中，持续压力变化是机械拉伸或变形的。然后，这些细胞启动了一个复杂的ECM营业额计划，以在几天的时间内调节出口电阻，并将IOP恢复到可接受的范围内。此外，出口围绕眼睛的圆圈高度分段，这对理解出口设施的所有方面和IOP稳态的机制产生了巨大的后果。我们提出的两个目的是揭开负责控制IOP稳态出口电阻调节的分子机制。这些研究将主要依靠TM和SC细胞培养，并灌注前节的器官培养。特定的目标1将需要在一系列时间点使用正常和青光眼的眼睛进行IOP稳态耐药性调节，在一系列时间点上，高流量区域的高流量区域与低流量区域的详细分子比较。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，西部免疫印迹和ELISA。具体目标2将是确定调节区域输出流动阻力变化的信号转导途径，这些变化响应于1倍至2倍压力变化。这将包括选择信号转导途径和转录调节器的关键组成部分的评估激活状态。验证参与IOP稳态过程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的遗传操纵以调节特定的途径成分，从而影响IOP稳态过程。这种详细的分子和细胞对IOP体内稳态过程的理解是在正常和青光眼的眼睛中如何调节的，这将提供新的目标，以恢复青光眼眼睛中的这种稳态过程。