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调查员(PI)提供必要的经验和技能，以成为青光眼研究领域的独立调查员。PI的博士和博士后研究都是在青光眼领域。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的小梁网和房水细胞生物学和生理学。他的特殊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，与活体猴子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的成像和小梁网络生物学方面的培训背景基础上再接再厉。他试图开发新的技术和实验方法来研究房水流出的可能调节机制。高眼压是青光眼的主要危险因素，但疾病过程中的错误导致眼压升高的原因尚不清楚。这项建议的长期目标是了解房水流出的调节。目前的科学焦点是寻求更好地理解眼内压(IOP)的可能调节机制。该提议的假设是，小梁网(TM)的收缩调节组织的流出阻力。目的1：建立和测试TM的收缩功能和流出阻力的检测方法；目标2：在合适的动物模型中研究TM的收缩功能和流出阻力。对于目标1，将建立使用灌流技术的流出阻力分析。下一步，包括组织形态计量学、免疫组织化学和Western blotting的收缩能力测试将被组装。这些检测将被用来评估TM在接触溶血磷脂酸(LPA)和转化生长因子-22(TGF22)后的收缩张力，这两种药物可以增强TM的收缩能力。RGS2纯合子敲除(RGS2-/-)小鼠具有收缩的血管表型和由于G蛋白信号改变而引起的高血压。这种损伤还会导致TM变得更具收缩能力。小鼠的眼压低于正常，这表明收缩张力的增加降低了流出阻力。对于AIM 2，将对RGS2-/-和野生型小鼠进行眼压、流出阻力和收缩能力测试。为了进一步改变收缩能力，并假定将其驱动到更高的状态，Caldesmon siRNA将通过前房输送到TM。在siRNA验证研究以确认沉默之后，将进行眼压、收缩和流出阻力分析。这种循序渐进的方法潜在地提供了对影响TM流出阻力的组织和分子调节机制的洞察。如果在小鼠体内成功传递siRNA，将为回答未来机制和治疗性质的问题提供合理的基础。这项调查将在南加州大学眼科进行。这里的眼科有着培养基础视力研究和临床科学家的深厚传统。拟议的研究将在Doheny Vision研究中心内的专用空间进行，该中心还容纳了研究所的核心设施，这些设施将支持PI的研究。PI将有相当多的受保护时间用于研究和教育计划。PI的研究和职业发展将在Sarah Hamm-Alvarez博士的指导下进行，Sarah Hamm-Alvarez博士是一名细胞和分子生物学家，在细胞骨架相互作用、相关的小鼠生物学、细胞成像以及药理学和药物输送方面拥有专业知识。PI将有Mark Humayun博士作为共同导师，他是一位临床科学家和生物工程师，在为眼睛和生物物理分析开发微电子系统方面拥有专业知识，这与研究小鼠房水流出系统的亚微流体学有关。保罗·考夫曼医学博士是活体动物房水生理学、眼部流出通路和青光眼治疗方面的专家，他将提供合作支持。该提案解决了一个与青光眼相关的研究问题。在研究、教学活动和支持性环境中的指导过程中，PI将获得宝贵的知识和技能，以发展作为独立研究人员和临床科学家的职业生涯。 公共卫生相关性：项目叙事青光眼是全球不可逆转失明的主要原因，其主要危险因素是眼压升高。这个项目研究了一种潜在的调节眼压的机制，可以帮助我们更好地了解青光眼及其治疗方法。

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.