Role of trabecular meshwork contractility in modulating outflow resistance

描述（由申请人提供）：本提案的总体目标是为首席研究员（PI）提供必要的经验和技能，使其成为青光眼研究领域的独立研究者。PI的博士和博士后研究方向为青光眼。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伤。他的博士后研究方向为青光眼的小梁网和房水细胞生物学和生理学。他的细胞生物学研究重点是细胞骨架和细胞-细胞外基质的相互作用，以及与活猴流体动力学研究中功能变化的相关性。他现在建议在青光眼的研究领域中建立他在成像和小梁网生物学方面的培训背景。他寻求发展新的技能和实验方法来研究假定的房水流出的调节机制。眼压升高是青光眼的主要危险因素，但在发病过程中出现什么问题导致眼压升高尚不清楚。这项提议的长期目标是了解房水流出调节。目前的科学重点是寻求更好地理解眼内压（IOP）的假设调节机制。该建议的假设是小梁网（TM）的收缩性调节组织的流出阻力。为了在活体小鼠中验证这一假设，我们提出了以下目的：目的1：建立并测试TM收缩性和流出阻力的测定方法；目的2：建立合适的动物模型，研究TM的收缩功能和流出阻力。对于Aim 1，将使用灌注技术建立流出阻力试验。接下来，将进行包括组织形态测定、免疫组织化学和免疫印迹在内的收缩性测定。这些试验将用于评估暴露于溶血磷脂酸（LPA）和转化生长因子-22 （TGF22）后的TM收缩张力，这些药物可增强TM收缩性。RGS2纯合子敲除（RGS2-/-）小鼠由于g蛋白信号的改变而具有血管收缩表型和高血压。这种损伤也会导致TM变得更容易收缩。小鼠的IOP低于正常水平表明，收缩张力的增加降低了流出阻力。在Aim 2中，将在RGS2-/-和野生型小鼠中进行IOP、流出阻力和收缩力测定。为了进一步改变收缩性并使其达到更高的状态，Caldesmon siRNA将通过前房传递到TM。在siRNA验证研究确认沉默后，将进行IOP、收缩性和流出阻力分析。这种循序渐进的方法有可能深入了解影响TM流出阻力的组织和分子调节机制。siRNA，如果成功地在小鼠体内传递，将为回答未来的机制和治疗性质问题提供一个合理的基础。这项调查将在南加州大学眼科部门进行。这里的眼科有着培养基础视觉研究和临床科学家的优良传统。拟议的研究将在Doheny视觉研究中心的专用空间进行，该中心还设有研究所的核心设施，将支持PI的研究。PI将有相当多的保护时间用于研究和教学教育计划。PI的研究和职业发展将在Sarah Hamm- Alvarez博士的指导下进行，她是一位细胞和分子生物学家，在细胞骨架相互作用、相关小鼠生物学、细胞成像、药理学和药物传递方面具有专业知识。该PI的共同导师Mark Humayun博士是一位临床科学家和生物工程师，他擅长开发用于眼睛和生物物理分析的微电子系统，这与研究小鼠水流出系统的亚微流体有关。Paul Kaufman医学博士是活体动物水生理、眼流出通道和青光眼治疗方面的专家，他将提供合作支持。该提案提出了一个与青光眼相关的研究问题。在研究过程中，教学活动，并在一个支持性的环境中指导，私家侦探将获得宝贵的知识和技能，发展成为一个独立的研究人员和临床科学家的职业生涯。

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.