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主要研究者（PI）提供成为青光眼研究领域独立研究者所需的经验和技能。 主要研究者的博士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伤。他的博士后研究方向是青光眼相关的小梁网和房水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-细胞外基质相互作用，与活猴流体动力学研究中的功能变化相关。他现在建议在青光眼研究领域内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一个假定的调节机制的房水流出。 眼压升高是青光眼的主要危险因素，但在疾病过程中发生什么错误导致眼压升高尚不清楚。这个提议的广泛的长期目标是了解房水流出调节。目前的科学焦点是寻求更好地理解眼内压（IOP）的假定调节机制。该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体小鼠中的假设：目的1：建立和测试TM收缩性和流出阻力的测定;目的2：在合适的动物模型中研究TM收缩功能和流出阻力。 对于目标1，将建立使用灌注技术的流出阻力测定。接下来，将组装包括组织形态计量学、免疫组织化学和蛋白质印迹的收缩性测定。这些试验将用于评价暴露于溶血磷脂酸（LPA）和转化生长因子-22（TGF-22）（增强TM收缩性的药物）后TM的收缩张力。 RGS 2纯合敲除（RGS 2-/-）小鼠具有收缩性血管表型和由于改变的G蛋白信号传导引起的高血压。这种损伤也会导致TM变得更加收缩。小鼠发展出比正常更低的IOP表明增加的收缩张力降低了流出阻力。 对于目标2，将在RGS 2-/-和野生型小鼠中进行IOP、流出阻力和收缩性试验。为了进一步改变收缩性并将其驱动到更高的状态，Caldesmon siRNA将通过前房递送到TM。在进行siRNA验证研究以确认沉默后，将进行IOP、收缩性和流出阻力测定。这种逐步的方法可能提供深入了解组织和分子调控机制影响TM的流出阻力。siRNA如果在小鼠体内成功递送，将为回答未来的机制和治疗性质的问题提供合理的基础。 本研究将在南加州大学眼科系进行。这里的眼科学系有着培养基础视觉研究和临床科学家的悠久传统。拟议的研究将在Doheny Vision Research Center内的专用空间进行，该中心还设有支持PI研究的研究所核心设施。PI将有相当多的研究时间和教学教育计划。 PI的研究和职业发展将在Sarah哈姆- Alvarez博士的指导下进行，Sarah是一名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、细胞成像以及药理学和药物输送方面具有专业知识。PI将有Mark Humayun博士作为共同导师，他是一名临床科学家和生物工程师，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。保罗考夫曼，医学博士，在活体动物水生理学，眼睛和青光眼治疗的流出途径的专家，将提供合作支持。 该提案解决了与青光眼相关的研究问题。在研究过程中，教学活动和支持性环境中的指导，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.