Aqueous humor dynamics studies in vivo and in vitro

描述（由申请人提供）：将研究降低眼压的新方法，眼压是青光眼进展的重要危险因素。通过小梁网的流出可能会被ILK， PI3K和Src激酶抑制剂增强，这些抑制剂已被证明可以改变培养的小梁网（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体外器官培养的前段和猴眼中进行研究。基因治疗方法将通过增强经巩膜途径的流出来降低IOP。目前最有效的青光眼药物治疗之一是基于前列腺素（PG）F2a的衍生物，它与FP受体结合，在一定程度上导致基质金属蛋白酶（MMP）的合成增强。这反过来又改变了睫状肌（CM）和巩膜细胞外环境的组成，导致巩膜流出增强。将携带PGF合成酶和基质溶解素（MMP-3）基因的慢病毒载体注射到活体猴眼前段，并评估其对IOP和巩膜流出的影响。PGF2a和MMP-3的产生也将在房水和体外转导的人TM和CM细胞培养基中进行评估。最后的目的是研究神经递质一氧化氮在体内调节眼内压、房水流入和流出中的作用。为了模拟青光眼前段一氧化氮合酶（NOS）水平的显著降低，在局部或眼内给药非选择性一氧化氮合酶抑制剂L-NAME和/或相对选择性神经元一氧化氮合酶抑制剂7-NI和/或NPLA后，在猴子体内产生短期等效的氮能去神经。对基本瞳孔直径、折射、房水形成和/或流出设施的影响将被确定。相反，这些参数将在是否存在胆碱能和/或肾上腺素能阻断的情况下对一氧化氮供体SNP的反应进行测量。这些结果将为开发降低IOP的新疗法提供重要的见解。研究将在活的非人灵长类动物中进行，在体外器官培养的前段中进行，在培养的小梁网细胞和睫状肌细胞中进行。将采用以下技术：通过荧光光度法形成房水；二级恒压灌注流出装置；由同位素稀释和富集测量计算的浮膜流出量；Goldmann眼压测量法；哈丁格重合折射法；通过游标卡尺测量瞳孔直径；通过定制设计的研究显微镜系统检测GFP荧光共表达在体内的基因表达；基因产物检测采用Western blots，酶免疫测定和免疫组织化学。公共卫生相关性：拟议的研究将探讨降低眼压的新方法，可能进一步发展用于青光眼治疗。前两个目标是通过使用新型小分子（PI3K， ILK和Src激酶抑制剂）增强房水通过两个已知的流出途径的流出，以增强小梁流出，以及基因治疗（慢病毒载体介导的基因传递，增加酶的产生（前列腺素合成酶和基质溶解素）），以增加巩膜流出。第三个目标是更好地了解一种重要的神经递质（一氧化氮）在调节正常和药物诱导的房水形成和引流变化中的作用，以便为新型青光眼治疗提供基础。

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.