The overall objective of this application is to determine the relationship between accelerated intracellular glycoxidation product formation in Muller cells and diabetic retinopathy.
Aim 1 is to examine in vitro the relationship between intracellular glycoxidation product formation and diabetes-induced Muller cell dysfunction. The effect of intracellular glycoxidation products on bcl-2 expressing Muller cell susceptibility to apoptosis will be investigated using quantitative immunoblotting to detect glyoxidation products and fluorescence-activated cell sorting and immunohistochemistry to detect chromatin condensation and DNA fragmentation. Effects on bcl-2 expression and in vivo binding to pro-apoptotic proteins will be evaluated in cell culture by RTPCR and quantitative immunoprecipitation. The effects of intracellular glycoxidation on VEGF and bFGF will be evaluated by RT-PCR, immunoprecipitation, immunoblotting, and mitogenic assays.
Aim 2 is to characterize the effect of selective reduction of the glycoxidation product precursors 3-deoxyglucosone and methylglyoxal on intracellular glycoxidation product levels. The activities of aldehyde reductase, aldose reductase, dihydrodiol dehydrogenase and glyoxalase I will be assessed in primary Muller cells. Cell lines transduced with highly efficient bicistronic HSV-1-derived vectors expressing both enzymes and a reporter gene will be assayed for 3-deoxyglucosone, methylglyoxal, and D-lactate, and glycoxidation products will be measured using quantitative immunoblotting.
Aim 3 is to generate transgenic mice that selectively express in Muller cells the enzymes that inhibit intracellular glyoxidation, using the p75 promoter. A bicistronic transgene construct will be used in which the internal ribosome entry site of EMC virus will allow co-expression of beta-galactosidase. Gene expression will be evaluated by X-gal histochemistry and by in situ hybridization. The number of apoptotoic cells in retinae of diabetic and non-diabetic transgenic and normal mice will be determined by the TUNEL technique, and the degree of diabetic retinopathy will be assessed by both RT-PCR of multiple basement membrane components and by retinal morphometry.
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