Molecular Mechanisms of SOD3 and Oxidative Stress in Diabetic Vitreoretinopathy

描述(申请人提供)：糖尿病玻璃体视网膜病变(DVR)是世界范围内日益严重的致盲原因。玻璃体内ROS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(SOD3)是人类玻璃体中一种重要的抗氧化防御酶，可能保护视网膜、睫状体和晶状体免受氧化损伤。我们建议的总体目标是研究SOD3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SOD3不同地定位于玻璃体亚结构，这些相互作用或SOD3酶活性的失调可能在DVR的病理生理学中起关键作用。项目目标包括：确定人玻璃体中SOD3蛋白/蛋白多糖相互作用的机制，确定SOD3糖基化在DVR中的功能影响，以及研究小鼠和人玻璃体中的SOD3氧化应激途径。将使用几种不同的技术。首先，蛋白质的相互作用将通过免疫共沉淀、蛋白质印迹、 用天然的、重组的和突变的SOD3进行免疫组织化学。接下来，我们将在体内和体外研究SOD3糖基化的功能效应。最后，将使用EPR光谱、免疫组织化学和Western blotting来测量人和小鼠DVR组织样本中SOD3、活性氧物种和氧化损伤的平衡。将对SOD3基因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情况。该项目对人类健康具有重要意义，因为了解眼睛中与SOD3相关的氧化应激途径有助于深入了解DVR的病理生理学机制，并提供一种独特的治疗方式。 公共卫生相关性：糖尿病玻璃体视网膜病变(DVR)是糖尿病的一种破坏性并发症，可能导致不可逆转的失明。该项目的目标是确定细胞外超氧化物歧化酶和氧化应激平衡如何影响DVR的病理。这项研究与美国国立卫生研究院的任务和NEI战略计划相关，因为它将在分子水平上增加对DVR发病机制的理解，并导致预防性治疗目标。

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. PUBLIC HEALTH RELEVANCE: Diabetic vitreoretinopathy (DVR) is a devastating complication of diabetes that may cause irreversible blindness. The goal of this project is to determine how extracellular superoxide dismutase and oxidative stress balance affects DVR pathology. This research is relevant to the NIH mission and NEI strategic plan because it will increase an understanding of DVR pathogenesis at a molecular level and lead to preventative therapeutic targets.