of work accomplished during the previous funding period involving the role of glucose derived nonenzymatic modifications of extracellular matrix in the pathogenesis of diabetic microvascular disease. It also provides preliminary data about the potential role of intracellular glycation in the pathogenesis of diabetic microvascular disease, and outlines in detail experiments to evaluate this unexplored mechanism. The hypothesis to be examined is that nonenzymatic glycation occurs intracellularly, where glucose derived sugars react with proteins up to 200 times faster than glucose. It is postulated that excessive intracellular glycation in vascular cells would modify the function of proteins involved in the regulation of normal cell growth and basement membrane production. bFGF has been selected as a model protein for intensive investigation (a) because it is stored intracellularly rather than secreted, and (b) because it plays an important role in microvascular homeostasis. To determine the effect of elevated extracellular glucose concentration on intracellular glycated protein and bFGF concentration, proteins from the endothelial cell line GM7373 will be analyzed using boronate affinity chromatography, Western blotting, and if required, immuno-PCR. To determine the effect of increased glucose 6-phosphate and other reactive glycolytic intermediates on intracellular glycation, expression vectors will be used to derive GM7373 endothelial cell clones stably transfected with the high Km beta-cell glucokinase gene. Clones will be characterized by immunoblotting, glucose 6-phosphate and glucokinase assays, and glycated proteins will be evaluated as described above. To evaluate the biologic consequences of intracellular glycation of bFGF, binding affinities will be evaluated using standard methods for radiolabelling, glycation, protein purification, and competitive radioreceptor assay. Characterization of glycated bFGF's biological activities will be assessed in standard mitogenesis and plasminogen activator generating assays. To characterize the specific chemical nature of intracellular glycation, amounts of intracellularly glycated bFGF required for analysis will be produced using CHO cells transfected with the amplifiable vector pEE14 containing bFGF cDNA. Peptide mapping, sequencing, and fast-atom bombardment mass spectroscopy will be used to analyze peptide and carbohydrate of intracellularly glycated bFGF.
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