Role of trabecular meshwork contractility in modulating outflow resistance

小梁网收缩力在调节流出阻力中的作用

• 批准号: 7953492
• 负责人: CHEE HIAN TAN
• 金额: $ 24.04万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-30 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7953492
• 关键词: Address; Affect; Animal Model; Animals; Aqueous Humor; Biological; Biological Assay; Biology; Biomedical Engineering; Blindness; Blood Vessels; California; Cells; Cellular biology; Clinical; Contracts; Core Facility; Cytoskeleton; Disease; Doctor of Philosophy; Drug Delivery Systems; Education; Environment; Exposure to; Extracellular Matrix; Eye; Fostering; Future; GTP-Binding Proteins; Gene Silencing; Glaucoma; Goals; Housing; Hypertension; Image; Immunohistochemistry; Impairment; Institutes; Investigation; Knock-out; Knockout Mice; Life; Liquid substance; Lysophospholipids; Measures; Mentors; Mentorship; Microfluidics; Molecular; Monkeys; Mus; Nature; Ophthalmology; Pathway interactions; Perfusion; Pharmacology; Phenotype; Physiologic Intraocular Pressure; Physiology; Principal Investigator; Process; RGS2 gene; Regulation; Relative (related person); Research; Research Personnel; Resistance; Risk Factors; Role; Scientist; Signal Transduction; Small Interfering RNA; System; Techniques; Testing; Therapeutic; Time; Tissues; Trabecular meshwork structure; Training; Transforming Growth Factors; Universities; Vision research; Western Blotting; Wild Type Mouse; abstracting; anterior chamber; aqueous; base; caldesmon; career; career development; cellular imaging; experience; in vivo; insight; lysophosphatidic acid; pressure; relating to nervous system; skills; validation studies

DESCRIPTION (provided by applicant): The overall goal of this proposal is to provide the principal investigator (PI) with the experience and skills necessary to become an independent investigator in the field of glaucoma research. The PI's Doctoral and Postdoctoral research were in the field of glaucoma. In his Doctoral research he developed in vivo quantitative confocal imaging approaches to study progressive neural damage. His postdoctoral research was in trabecular meshwork and aqueous humor cell biology and physiology with respect to glaucoma. His specific cell biological focus was in cytoskeleton and cell- extracellular matrix interactions, with correlation to functional changes in live monkey hydrodynamic studies. He now proposes to build on his training background in imaging and trabecular meshwork biology within the research field of glaucoma. He seeks to develop new skills and experimental approaches to study a putative regulatory mechanism of aqueous humor outflow. Elevated IOP is the major risk factor for glaucoma but what goes wrong in the disease process to cause IOP elevation is unknown. The broad long-term goal of this proposal is to understand aqueous humor outflow regulation. The present scientific focus is to seek to better understand a putative regulatory mechanism for intraocular pressure (IOP). The proposal's hypothesis is that contractility of the trabecular meshwork (TM) modulates the outflow resistance of the tissue. The following aims are proposed to address the hypothesis in the live mouse: Aim 1: Establish and test assays for TM contractility and outflow resistance; Aim 2: Study TM contractile function and outflow resistance in a suitable animal model. For Aim 1, an assay for outflow resistance using perfusion techniques will be established. Next a contractility assay involving histomorphometry, immunohistochemistry and Western blotting will be assembled. These assays will be used to evaluate the TM's contractile tone after exposure to lysophosphatidic acid (LPA) and transforming growth factor-22 (TGF22), agents that enhance TM contractility. The RGS2 homozygous knockout (RGS2-/-) mouse has a contractile vascular phenotype and hypertension due to altered G-protein signaling. This impairment also causes the TM to become more contractile. That the mouse develops a lower IOP than normal suggests that the increased contractile tone decreases outflow resistance. For Aim 2, IOP, outflow resistance and contractility assays will be performed in RGS2-/- and wild- type mice. To alter contractility further and putatively drive it to a more heightened state, Caldesmon siRNA will be delivered to the TM via the anterior chamber. After siRNA validation studies to confirm silencing, IOP, contractility and outflow resistance assays will be performed. This stepwise approach potentially provides insights into tissue and molecular regulatory mechanisms affecting the TM's outflow resistance. siRNA, if successfully delivered in the mouse in vivo, will provide a rational basis for answering future questions of both mechanistic and therapeutic nature. This investigation will be based at the Department of Ophthalmology of the University of Southern California. The Department of Ophthalmology here has a strong tradition of fostering basic vision research and clinical scientists. The proposed research will be conducted in dedicated space within the Doheny Vision Research Center, which also houses the Institute's Core facilities that will support the PI's research. The PI will have considerable protected time for research and a plan of didactic education. The PI's research and career development will proceed under the mentorship of Sarah Hamm- Alvarez, PhD, a cell and molecular biologist with expertise in cytoskeleton interactions, related mouse biology, cellular imaging, and pharmacology and drug delivery. The PI will have as a co-mentor Mark Humayun, MD PhD, a clinician scientist and bioengineer with expertise in developing microelectronic systems for the eye and biophysical analysis, which is pertinent to studying the sub-microfluidics of the mouse aqueous outflow system. Paul Kaufman, MD, an expert in live animal aqueous physiology, the outflow pathways of the eye and glaucoma therapies, will provide collaborative support. The proposal addresses a research question of relevance to glaucoma. In the course of the research, didactic activities, and mentorship within a supportive environment, the PI will gain invaluable knowhow and skills for developing a career as an independent researcher and clinical scientist. PUBLIC HEALTH RELEVANCE: Project narrative Glaucoma, the leading cause of irreversible blindness worldwide, has as its major risk factor elevated intraocular pressure. This project studies a potential mechanism for regulating intraocular pressure that can help us better understand glaucoma and ways to treat it.

描述（由申请人提供）：该提案的总体目标是向主要研究者（PI）提供成为青光眼研究领域的独立研究者所必需的经验和技能。 PI的博士学位和博士后研究是在青光眼领域。在他的博士研究中，他开发了体内定量共聚焦成像方法来研究进行性神经损害。他的博士后研究是针对小梁网术和有关青光眼的水性幽默细胞生物学和生理学。他的特定细胞生物学重点是细胞骨架和细胞外基质相互作用，与活猴流体动力学研究的功能变化相关。他现在建议在青光眼研究领域内的成像和小梁网络生物学领域的训练背景。他试图开发新的技能和实验方法，以研究水性幽默流出的推定调节机制。 IOP升高是青光眼的主要危险因素，但是疾病过程中出现了IOP升高的问题是未知的。该提案的长期长期目标是了解水性幽默流出调节。目前的科学重点是寻求更好地了解眼内压力（IOP）的推定调节机制。提案的假设是小梁网（TM）的收缩性调节组织的流出阻力。 提出了以下目的来解决活小鼠中的假设：目标1：建立和测试TM收缩性和耐药性的测定； AIM 2：在合适的动物模型中研究TM收缩功能和流出阻力。 对于AIM 1，将建立使用灌注技术的耐药性测定法。接下来，将组装涉及组织形态学，免疫组织化学和蛋白质印迹的收缩性测定。这些测定将用于评估暴露于溶血磷脂酸（LPA）和转化生长因子22（TGF22）后TM的收缩张力，这是增强TM收缩力的药物。 RGS2纯合子敲除（RGS2 - / - ）小鼠具有收缩的血管表型和由于G蛋白信号的改变而导致的高血压。这种障碍还导致TM变得更加收缩。小鼠发育的IOP比正常水平低，这表明收缩音的增加会降低流出电阻。 对于AIM 2，将在RGS2 - / - 和野生型小鼠中进行IOP，流出性和收缩性测定。为了进一步改变收缩力并将其推动到更高的状态，Caldesmon sirna将通过前室交付到TM。在确认沉默的siRNA验证研究之后，将进行IOP，收缩力和流出性测定法。这种逐步方法有可能提供有关影响TM流出抗性的组织和分子调节机制的见解。 siRNA如果在体内鼠标中成功传递，将为回答机械性和治疗性质的未来问题提供合理的基础。 这项调查将基于南加州大学眼科系。这里的眼科系具有促进基本视觉研究和临床科学家的扎实传统。拟议的研究将在Doheny Vision Research Center的专用空间中进行，该研究中心还设有该研究所的核心设施，以支持PI的研究。 PI将有大量的研究时间和教育计划。 PI的研究和职业发展将在Sarah Hamm-Alvarez博士的指导下进行，该博士是细胞和分子生物学家，具有细胞骨架相互作用，相关的小鼠生物学，细胞成像以及药理学和药物输送方面的专业知识。 PI将作为临床医生和生物工程师Mark Humayun，MD Humayun，具有为眼睛和生物物理分析开发的微电体系统的专业知识，这与研究小鼠水流流出系统的亚微富集学有关。保罗·考夫曼（Paul Kaufman），医学博士，现场动物水性生理学专家，眼睛和青光眼疗法的流出途径，将提供协作支持。 该提案解决了与青光眼相关的研究问题。在研究，教学活动和支持环境中的指导过程中，PI将获得宝贵的专业知识和技能，以发展作为独立研究人员和临床科学家的职业。 公共卫生相关性：全球不可逆失明的主要原因项目叙事青光眼是其主要危险因素升高的眼内压力。该项目研究了调节眼压的潜在机制，可以帮助我们更好地理解青光眼及其治疗方法。