The overall goal of this application is to find markers of early changes in diabetes that will predict severity of later disease so that patients can be appropriately monitored and treated. The most convenient markers are those measured in blood. Therefore we propose to search for early surrogate markers that might be found in serum. Loss of retinal vascular cells is a salient early feature that leads to diabetic retinopathy, and probably reflects vascular changes that occur throughout the body. Thus soluble proteins associated with microvascular cell death may become a useful surrogate end-point in experimental diabetic retinopathy as well as early markers for other clinical changes that lead to end stage disease. It is not practical to search for changes in gene expression in diabetic animal models because in these models, the early vascular changes in the retina are not well characterized, are variable, and are prolonged. Therefore we need a model with rapid and profound loss of vessels, gene expression changes are amplified. In peritnatal mice, hyperoxia inhibits development of retinal blood vessels and causes greater than 80% vessel regression over 5 days through apoptosis, a process that is likely to parallel early stage diabetic retinopathy. The rapid and amplified vessel loss in hyperoxia provides a good model to study the underlying biochemical mechanisms involved in retinal vessel degeneration. Gene chip array technique will be used to assess difference in gene expression between hyperoxia treated and normal mice at 4 time points. Changes over time in each group will also be assessed. Information will be analyzed using 4 different approaches to sort the genes of secreted factors. The top 20 candidates will be verified by TaqMan real-time RT-PCR to assess copy number expressed per control gene (cyclophilin). The soluble proteins of the best candidates will be assessed in vitro in retinal pericyte and endothelial cells to be certain that they are associated with apoptosis and vessel loss. Using these approaches we are likely to identify gene changes leading to soluble protein factors associated with diabetic retinal and other vessel loss. These factors may be used as surrogate markers for effective early detection of diabetic changes through routine screening of patients' blood or may lead to new therapeutic interventions aimed at blocking the deleterious actions of those factors.
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