Advanced glycation and products are found in increased amounts in extracellular structures of diabetic retinal vessels (42-44) and renal glomeruli (45-47). These AGEs were originally thought to arise from nonenzymatic reactions between extracellular proteins and glucose. However, the rate of AGE formation from glucose is orders of magnitude slower than the rate of AGE formation from glucose-derived dicarbonyl precursors generated intracellularly, and it now seems likely that intracellular hyper-glycemia is the primary initiating event in the formation of both intracellular and extracellular AGEs (48). AGEs can arise from intracellular autoxidation of glucose to glyoxal (49), decomposition of the Amadori product to 3-deoxyglucosone (perhaps accelerated by an amadoriase), and fragmentation of glyceraldehyde-3-phosphate to methylglyoxal (50) (see Fig. 3). These reactive intracellular dicarbonyls react with amino groups of intracellular and extracellular proteins to form AGEs. Methylglyoxal and glyoxal are detoxified by the glyoxalase system (50).
Intracellular production of AGE precursors damages target cells by three general mechanisms. First, intracellular proteins modified by AGEs have altered function. Second, extracellular matrix components modified by AGE precursors interact abnormally with other matrix components and with matrix receptors (integrins) on cells. Third,
plasma proteins modified by AGE precursors bind to AGE receptors on cells such as macrophages, inducing receptor-mediated reactive oxygen species production. This AGE receptor ligation activates the pleiotrophic transcription factor NF-kB, causing pathologic changes in gene expression (51).
In endothelial cells, intracellular AGE formation occurs very quickly. Proteins involved in macromolecular endocytosis are modified by AGEs because the 2.2-fold increase in endocytosis induced by hyperglycemia is also prevented by overexpression of the methylglyoxal-detoxifying glyoxalase I (52). Glyoxalase-I overexpression also completely prevents the fourfold hyperglycemia-induced increase in Muller cell expression of angiopoietin-2, a factor that has been implicated in both pericyte loss and capillary regression (53-55).
Intracellular AGEs leak out of cells and alter the functional properties of several important matrix molecules. Collagen was the first matrix protein used to demonstrate that glucose-derived AGEs form covalent, intermolecular bonds. In vitro AGE formation on intact glomerular basement membrane increases its permeability to albumin in a manner that resembles the abnormal permeability of diabetic nephropathy (56,57). AGE formation on extracellular matrix not only interferes with matrix-matrix interactions, it also interferes with matrix-cell interactions. For example, AGE modification of type IV collagen's cell-binding domains decrease endothelial cell adhesion (58), and AGE modification of a six-amino-acid growth-promoting sequence in the A-chain of the laminin molecule markedly reduces neurite outgrowth (59). AGE modification of vitronectin reduced cell attachment-promoting activity (60).
Specific receptors for AGEs were first identified on monocytes and macrophages. Two AGE-binding proteins isolated from rat liver are both present on monocyte/macrophages. Antisera to either the 60-kDa or 90-kDa protein, recently identified as OST-48 and 80K-H, respectively (61), block AGE binding (62). AGE protein binding to this receptor stimulates macrophage production of IL-1, insulin-like growth factor-1 (IGF-1), TNF-a, TGF-P, macrophage colony-stimulating factor and, granulocyte/macrophage colony-stimulating factor at levels that have been shown to increase glomerular synthesis of type IV collagen and to stimulate proliferation and chemotaxis of both arterial smooth muscle cells and macrophages (33-35,63-68). The macrophage scavenger receptor type II and galectin-3 have also been shown to recognize AGEs (69-72).
Vascular endothelial cells also express AGE-specific receptors (RAGEs). A 35-kDa and a 46-kDa AGE-binding protein have been purified to homogeneity from endothe-lial cells (73-75).
In endothelial cells, AGE binding to its receptor induces changes in gene expression that include alterations in thrombomodulin, tissue factor, and VCAM-1 (25-28). These changes induce procoagulatory changes in the endothelial surface and increase the adhesion of inflammatory cells to the vessel wall. In addition, endothelial AGE-receptor-binding appears to mediate, in part, the hyperpermeability induced by diabetes, probably through the induction of vascular endothelial growth factor (VEGF) (76-78).
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