Aqueous humor dynamics studies in vivo and in vitro

体内和体外房水动力学研究

基本信息

批准号：
7895519
负责人：
PAUL L KAUFMAN
金额：
$ 37.11万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-08-01 至 2012-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7895519
关键词：
Abbreviations Adrenergic Agents Anterior Aqueous Humor Binding Biological Assay Caliber Cell Adhesion Cells Ciliary Muscle Custom Cytoskeleton Denervation Detection Dose Drainage procedure Environment Enzyme Immunoassay Enzymes Extracellular Matrix Eye Fluorescence Fluorophotometry Functional disorder Gene Delivery Gene Expression Genes Glaucoma Glucocorticoids Goals Green Fluorescent Proteins Human Immunohistochemistry In Vitro Intercellular Junctions Intramuscular Intravenous Isotopes Lentivirus Vector Life Link Long-Term Effects Macaca fascicularis Matrix Metalloproteinases Measures Mediating Microscope Monkeys Muscle Cells NG-Nitroarginine Methyl Ester Neurotransmitters Nitric Oxide Nitric Oxide Donors Nitric Oxide Synthase Nitric Oxide Synthase Type I Organ Organ Culture Techniques PGF receptor PTGS2 gene Pathway interactions Perfusion Pharmaceutical Preparations Pharmacotherapy Phosphotransferases Physiologic Intraocular Pressure Production Prostaglandin-Endoperoxide Synthase Prostaglandins Proteins Pupil Refractometry Regulation Research Design Risk Factors Role Sclera Simulate Stromelysin 1 System Techniques Time Tissue Inhibitor of Metalloproteinases Trabecular meshwork structure Transgenes Viral Vector Western Blotting adrenergic base cholinergic extracellular gene therapy in vivo inhibitor/antagonist insight integrin-linked kinase kinase inhibitor myocilin nerve supply nonhuman primate novel novel strategies novel therapeutic intervention pressure prostaglandin-F synthase public health relevance response small molecule src-Family Kinases tonometry transgene expression

项目摘要

DESCRIPTION (provided by applicant): New approaches to lower intraocular pressure, a significant risk factor for the progression of glaucoma, will be investigated. Outflow through the trabecular meshwork will potentially be enhanced by ILK, PI3K and Src kinase inhibitors, which have been shown to alter the cytoskeleton and cellular adhesions of trabecular meshwork (TM) cells in culture. The effects of these compounds on intraocular pressure (IOP) and outflow facility will be studied in organ-cultured anterior segments and in monkey eyes in vivo. Gene therapy approaches will be utilized to lower IOP by enhancing outflow via the uveoscleral pathway. One of the current most effective pharmacotherapies for glaucoma is based on derivatives of prostaglandin (PG)F2a which binds to the FP receptor leading, in part, to enhanced matrix metalloproteinase (MMP) synthesis. This, in turn, alters the composition of the extracellular environment of the ciliary muscle (CM) and sclera leading to an enhancement of uveoscleral outflow. Lentiviral vectors carrying genes for PGF synthase and for stromelysin (MMP-3) will be injected into the anterior segment of monkey eyes in vivo and the effects on IOP and uveoscleral outflow will be assessed. PGF2a and MMP-3 production will also be assessed in the aqueous humor and in the media from human TM and CM cells transduced in vitro. A final objective will be to investigate in vivo the role of the neurotransmitter, nitric oxide, in regulating IOP, aqueous humor inflow and outflow. To simulate the dramatically reduced levels of nitric oxide synthase (NOS) in the anterior segment in glaucoma, a short-term equivalent of nitrergic denervation will be created in monkeys in vivo following topical or intracameral dosing with the non-selective NOS inhibitor L-NAME and/or the relatively selective neuronal nitric oxide synthase inhibitors 7-NI and/or NPLA. The effects on basal pupil diameter, refraction, aqueous humor formation and/or outflow facility will be determined. Conversely, these parameters will be measured in response to the nitric oxide donor, SNP, in the presence and absence of cholinergic and/or adrenergic blockade. These results will provide important insights for developing new therapies to lower IOP. Studies will be conducted in living nonhuman primates, in vitro in organ-cultured anterior segments and in trabecular meshwork cells and ciliary muscle cells in culture. The following techniques will be employed: aqueous humor formation by fluorophotometry; outflow facility by two-level constant pressure perfusion; uveoscleral outflow calculated from isotope dilution and accumulation measures; IOP by Goldmann applanation tonometry; refraction by Hartinger coincidence refractometry; pupil diameter via vernier calipers; gene expression in vivo by coexpression of GFP fluorescence detected by a custom designed research microscope system; gene product detection by Western blots and enzyme immunoassays and immunohistochemistry. PUBLIC HEALTH RELEVANCE: The proposed studies will investigate new approaches to lower intraocular pressure that may be further developed for glaucoma therapy. The first two objectives are to enhance aqueous humor outflow through the two known outflow pathways using novel small molecules (PI3K, ILK and Src kinase inhibitors) to enhance trabecular outflow, and gene therapy (lentiviral vector mediated delivery of genes that increase production of enzymes (prostaglandin synthase and stromelysin)) to increase uveoscleral outflow. A third goal is to better understand the role of an important neurotransmitter (nitric oxide) in regulating normal and drug induced changes in aqueous humor formation and drainage in order to provide the basis for novel glaucoma therapies.

描述（由申请人提供）：将研究降低眼内压的新方法，这是青光眼发展的重要危险因素。通过小梁，PI3K和SRC激酶抑制剂可以增强穿过小梁网的流出，这些抑制剂已证明会改变培养物中小梁网（TM）细胞的细胞骨架和细胞粘附。这些化合物对眼内压（IOP）和流出设施的影响将在器官培养的前部和体内猴眼中进行研究。基因治疗方法将通过葡萄球菌途径增强流出，以降低IOP。目前最有效的青光眼药物疗法之一是基于前列腺素（PG）F2A的衍生物，该衍生物与FP受体结合，部分地与增强的基质基质金属蛋白酶（MMP）合成。反过来，这改变了睫状肌（CM）和巩膜的细胞外环境的组成，从而增强了葡萄球菌流出。携带PGF合酶基因和Stromelysin（MMP-3）的慢病毒载体将被注入体内猴眼前部分，并评估对IOP和葡萄球菌流出的影响。 PGF2A和MMP-3的产生也将在水性幽默和人类TM和CM细胞的培养基中进行评估。最终的目标是在体内研究神经递质一氧化氮在调节IOP，水性幽默流入和流出中的作用。为了模拟青光眼前节中的一氧化氮合酶（NOS）的大幅度降低水平NPLA。将确定对基底瞳孔直径，折射，水性幽默形成和/或流出设施的影响。相反，在存在和不存在胆碱能和/或肾上腺素能阻断的情况下，将根据一氧化氮供体SNP进行测量这些参数。这些结果将为开发新疗法以降低IOP提供重要见解。研究将在生存的非人类灵长类动物，在器官培养的前部以及小梁网状细胞和培养中纤毛肌肉细胞中进行研究。将采用以下技术：通过荧光测定法形成水性幽默；通过两级恒压灌注流出设施；根据同位素稀释和积累措施计算出的葡萄球菌流出； IOP撰写的Goldmann Applanation Tonometry； Hartinger重合折射率折射；通过游标卡尺的学生直径；通过自定义设计的研究显微镜系统检测到的GFP荧光的GFP荧光的体内基因表达；通过蛋白质印迹和酶免疫测定和免疫组织化学检测基因产物。公共卫生相关性：拟议的研究将研究降低眼内压力的新方法，这可能进一步用于青光眼治疗。前两个目标是使用新型的小分子（PI3K，ILK和SRC激酶抑制剂）通过两种已知的流出途径增强水性幽默流出，以增强小梁疗法（慢病毒载体介导的基因介导的基因递送）增加了enzymes Outsypase anfformist anfforpage ands inthlape and consyntaly synthalsy synthanpase and synthalsy synthanpase and synthanpase synthanpase and synthanpase and consys synthanpase synthanpase synthanpers ande tormsys ande tormess ande疗法。第三个目标是更好地了解重要的神经递质（一氧化氮）在调节正常和药物诱导的幽默形成和排水的变化中的作用，以便为新型的青光眼治疗提供基础。