Molecular Mechanisms of SOD3 and Oxidative Stress in Diabetic Vitreoretinopathy

描述（由申请人提供）：糖尿病性玻璃体视网膜病变（DVR）是全球范围内日益增长的致盲原因。玻璃体中活性氧（ROS）的升高与DVR的发病机制有关，但其分子机制尚不清楚。我们的研究表明，细胞外超氧化物歧化酶（SOD 3）是一种重要的抗氧化防御酶在人类玻璃体，并可能保护视网膜，睫状体，和透镜免受氧化损伤。我们的总体目标是研究SOD 3在正常和糖尿病玻璃体中的分子机制和功能。我们的中心假设是，SOD 3差异定位于玻璃体亚结构和失调，这些相互作用或SOD 3酶活性可能是至关重要的DVR的病理生理。项目目标包括：鉴定人玻璃体中的SOD 3蛋白/蛋白聚糖相互作用机制，确定DVR中SOD 3糖基化的功能作用，并研究小鼠和人玻璃体中的SOD 3氧化应激途径。将使用几种不同的技术。首先，蛋白质相互作用将通过共免疫沉淀，蛋白质印迹， 和使用天然、重组和突变SOD 3的免疫组织化学。接下来，将在体内和体外研究SOD 3糖基化的功能作用。最后，将使用EPR光谱法、免疫组织化学和蛋白质印迹法在人和小鼠DVR组织样品中测量SOD 3、活性氧和氧化损伤的平衡。将对SOD 3敲除小鼠进行详细的临床表型分析，并确定视网膜中氧化应激途径的激活。该项目对人类健康具有重要意义，因为了解眼睛中SOD 3相关的氧化应激途径可以深入了解DVR病理生理学机制，并提供独特的治疗方式。

DESCRIPTION (provided by applicant): Diabetic vitreoretinopathy (DVR) is a growing cause of blindness across the world. Elevation of intraocular reactive oxygen species (ROS) in the vitreous is implicated in the pathogenesis of DVR, but the molecular mechanisms are not known. Our studies suggest extracellular superoxide dismutase (SOD3) is an important antioxidant defense enzyme in the human vitreous and may protect the retina, ciliary body, and lens from oxidative damage. The overall objective of our proposal is to investigate the molecular mechanisms and function of SOD3 in the normal and diabetic vitreous. Our central hypothesis is that SOD3 is differentially localized to vitreous substructures and dysregulation of these interactions or SOD3 enzymatic activity may be critical in the pathophysiology of DVR. Project goals include: identifying SOD3 protein/proteoglycan interaction mechanisms in the human vitreous, determining the functional effects of SOD3 glycation in DVR, and investigating SOD3 oxidative stress pathways in the mouse and human vitreous. Several different techniques will be utilized. First, protein interactions will be assessed by co-immunoprecipitation, western blotting, and immunohistochemistry using native, recombinant, and mutant SOD3. Next, the functional effects of SOD3 glycation will be studied in vivo and in vitro. Finally, the balance of SOD3, reactive oxygen species, and oxidative damage will be measured in human and mouse DVR tissue samples using EPR spectroscopy, immunohistochemistry and western blotting. Detailed clinical phenotyping of SOD3 knockout mice will be performed and activation of oxidative stress pathways in the retina will be determined. The project is significant to human health because understanding SOD3-related oxidative stress pathways in the eye gives insight into the mechanisms of DVR pathophysiology and provides a unique therapeutic modality.