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.