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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
2R01DK033861-09A1
Application #
3232259
Study Section
Pathology A Study Section (PTHA)
Project Start
1984-04-01
Project End
1996-07-31
Budget Start
1993-08-01
Budget End
1994-07-31
Support Year
9
Fiscal Year
1993
Total Cost
Indirect Cost
Name
Albert Einstein College of Medicine
Department
Type
Schools of Medicine
DUNS #
009095365
City
Bronx
State
NY
Country
United States
Zip Code
10461
Giacco, Ferdinando; Du, Xueliang; CarratĂș, Anna et al. (2015) GLP-1 Cleavage Product Reverses Persistent ROS Generation After Transient Hyperglycemia by Disrupting an ROS-Generating Feedback Loop. Diabetes 64:3273-84
Giacco, Ferdinando; Du, Xueliang; D'Agati, Vivette D et al. (2014) Knockdown of glyoxalase 1 mimics diabetic nephropathy in nondiabetic mice. Diabetes 63:291-9
Golovchenko, I; Goalstone, M L; Watson, P et al. (2000) Hyperinsulinemia enhances transcriptional activity of nuclear factor-kappaB induced by angiotensin II, hyperglycemia, and advanced glycosylation end products in vascular smooth muscle cells. Circ Res 87:746-52
Nishikawa, T; Giardino, I; Edelstein, D et al. (2000) Changes in diabetic retinal matrix protein mRNA levels in a common transgenic mouse strain. Curr Eye Res 21:581-7
Du, X L; Edelstein, D; Rossetti, L et al. (2000) Hyperglycemia-induced mitochondrial superoxide overproduction activates the hexosamine pathway and induces plasminogen activator inhibitor-1 expression by increasing Sp1 glycosylation. Proc Natl Acad Sci U S A 97:12222-6
Hammes, H P; Brownlee, M; Lin, J et al. (1999) Diabetic retinopathy risk correlates with intracellular concentrations of the glycoxidation product Nepsilon-(carboxymethyl) lysine independently of glycohaemoglobin concentrations. Diabetologia 42:603-7
Shinohara, M; Thornalley, P J; Giardino, I et al. (1998) Overexpression of glyoxalase-I in bovine endothelial cells inhibits intracellular advanced glycation endproduct formation and prevents hyperglycemia-induced increases in macromolecular endocytosis. J Clin Invest 101:1142-7
Giardino, I; Fard, A K; Hatchell, D L et al. (1998) Aminoguanidine inhibits reactive oxygen species formation, lipid peroxidation, and oxidant-induced apoptosis. Diabetes 47:1114-20
Hammes, H P; Brownlee, M; Jonczyk, A et al. (1996) Subcutaneous injection of a cyclic peptide antagonist of vitronectin receptor-type integrins inhibits retinal neovascularization. Nat Med 2:529-33
Giardino, I; Edelstein, D; Brownlee, M (1996) BCL-2 expression or antioxidants prevent hyperglycemia-induced formation of intracellular advanced glycation endproducts in bovine endothelial cells. J Clin Invest 97:1422-8

Showing the most recent 10 out of 15 publications