A. Advanced Glycation End Products
Advanced glycation end products (AGEs) are a heterogeneous group of irreversible adducts resulting from nonenzymatic glycation and oxidation of proteins, lipids, and nucleic acids. Glucose and other reducing sugars react in a nonenzymatic reaction (Maillard reaction) with the N-terminal residues and/ or e-amino groups of proteins initially forming a Schiff base. Rearrangement of this aldimine leads after a short time to the formation of more stable but still reversible Amadori adducts. The open chain of the resulting ketoamin can react with other amino groups. Oxidation, dehydration, and condensation reactions finally lead to the production of irreversible crosslinks, which are proteinase resistant.
The formation of AGEs in vitro and in vivo depends on the turnover rate of the modified substrate, sugar concentration, and time. Recent studies have shown that AGEs can be formed not only at long-living proteins but occur also on short-living proteins (48), peptides (48), lipids (49), and nucleic acids (50-52).
AGE formation and protein crosslinking alter the structural and functional properties of proteins, lipid components, and nucleic acids. AGEs have also been shown to induce cellular signaling, activation of transcription factors, and consequently gene expression in vitro and in vivo (32). They have been suggested to represent general markers of oxidative stress and long-term damage to proteins and to induce pathogenic changes in endothelial cells. Thus, AGEs are not only markers but also mediators of chronic vasuclar diseases and late diabetic complications.
AGE formation proceeds slowly under normal glycemic conditions but is enhanced in the presence of hyperglycemia, oxidative stress, and/or conditions in which protein and lipid turnover are prolonged. For example, /V-epsilon-(carboxymethyl)lysine (CML), one of the various AGE structures postulated to date, has been found to be a product of both glycoxidation (combined non-enzymatic glycation and oxidation) and lipid peroxidation reactions (53). CML and pentosidine have been shown to accumulate in diabetic kidneys in colocal-ization with a marker of lipid peroxidation (MDA), suggesting an association of local oxidative stress with the etiology of diabetic glomerular lesions (54). Evidence for an age-dependent increase in CML accumulation in distinct localizations and acceleration of this process in diabetes has been provided by immunolocalization of CML in skin, lung, heart, kidney, intestine, intervertebral discs, and particularly in arteries (55). In diabetic kidneys, AGEs were preferentially localized in vascular lesions (56), renal cortex (57), expanded mesangial areas (58), and the glomerular basement membrane (56-59). An increased CML content in serum proteins of diabetic patients (55) and a correlation of serum AGE levels with the progressive loss of kidney function was found (60). The increased formation of tissue AGEs has been described to precede and to correlate with early manifestations of renal and retinal complications in patients with diabetes (61).
Increased levels of AGE-modified low-density lipoprotein (LDL) with a markedly impaired clearance have been found in the plasma of diabetic patients, suggesting a pathway for pathogenic modification of LDL (62). The mediating role of AGEs in development of late diabetic complications (Table 1) (49,63-70) has been studied in animal models by short- and long-term administration of AGEs. Short-term administration of AGEs led to increased vascular permeability and leakage, impaired endothelial relaxation, subendo-thelial mononuclear recruitment, activation of NF-kB, and subsequent VCAM-1 gene expression (71-73). Long-term administration of AGEs resulted in arteriolar basement thickening and complex vascular dysfunction (74) and in glomerular basement thickening, mesangial expansion, glomerulosclerosis, and proteinuria (68).
The principal means through which AGEs exert their cellular effects is via specific cellular receptors (Table 2). One of them, the receptor for AGE (RAGE), a 35-kDa protein, is also expressed by endothelial cells (23,75,76).
Formation of HbAk as a marker of production of AGEs: poor glycemic control increases formation of AGEs and AGE-dependent cell activation (37,41,63) Toxic effects of AGEs on retinal endothelial cells (64) and positive correlation between accumulation of AGEs, expression of vascular endothelial growth factor, and nonproliferative and proliferative diabetic retinopathy (65) Inhibition of development of experimental diabetic retinopathy by aminoguanidine treatment (49).
Excessive deposition of intra- and extracellular AGEs in human diabetic peripheral nerve (66)
Inhibition of AGE formation prevents diabetic peripheral nerve dysfunction (67) Accumulation of AGEs in the kidney of diabetic patients (56,60) Injection of AGE-albumin in normal rats induces symptoms of diabetic nephropathy (68)
Blocking of AGE binding to RAGE reduces albuminuria (69) Inhibitor AGEs reduces urinary ablumin excretion, mesangial expansion, and glomerular basement membrane thickening (70)
Table 2 AGE Binding Proteins and Their Localization
AGE binding proteins AGE-R, (OST-48)
AGE-R, (Galectin-3 or GBP-35) RAGE
Lactoferrin, lysozyme Fructosylline-specific binding protein Macrophage scavenger receptor
Monocytes/macrophages, endothelial cells, T lymphocytes, mesangial cells, neurons Monocytes/macrophages, endothelial cells, T lymphocytes, fibroblasts, mesangial cells, neurons Monocytes/macrophages, endothelial cells,
T lymphocytes, Endothelial cells, monocytes/ macrophages, smooth muscle cells, mesangial cells, neurons, T lymphocytes, erythrocytes Endothelial cells Monocytes Macrophages
An induction of endothelial RAGE expression has been shown on vessels from patients with arteriosclerosis, diabetes, uremia, and vasculitis (77-79). Binding of AGEs to their cellular binding sites results in generation of oxygen free radicals and depletion of antioxidants such as glutathione and ascorbate (32,80). The consequently enhanced cellular oxidative stress leads to activation of the redox-sensitive transcription factor NF-kB in endothelial cells, smooth muscle cells, mesangial cells, and monocytes/macrophages (23,25,32,78-81).
The multiprotein complex NF-kB resides as an inactive form in the cytoplasm associated with its inhibitor, IkB (Table 3). NF-kB translocates to the nucleus after phosphorylation and proteolitic degradation of IkB. NF-kB activation is modulated by redox reactions that increase the cytosolic phosphorylation, and degradation of IkB and requires a thioredoxin-dependent status in the nucleus (82-85). NF-KB-dependent genes and their products [include] iKBa, RAGE, cytokines (tumor necrosis factor-a, interleukin-6 and -8), adhesion molecules (VCAM-1, ICAM-1, ELAM), receptors for coagulation factors such as the procoagulant tissue factor endothelin-1, inducible nitric oxide synthase, inducible cyclooxygenase, heme oxygenase type 1, and 5-lipoxygenase (23,77,86). Because transcription of iKBa is autoregulated by NF-kB (87), activation of NF-kB terminates itself (86,88), leading to a short-living acute cellular response. Recent studies showed that IkB|3 mediates a more sustained activation of NF-kB that lasts up to 48 h (89,90).
Activation of NF-kB and induction of increased binding activity of NF-kB are believed to have a pivotal role in the pathogenesis and progression of chronic diseases, such as diabetes and atherosclerosis (39,86,91,92). Accumu-
Table 3 Proteins of the NF-kB and IkB Families
Proteins of the NF-kB family Proteins of the IkB family
P50 (pi50) IkBoc
P52/p49 (pi00) IkBP
P65 (relA) ikny c-rel IkBe relB IkB-R
lating data indicate a close link between hyperglycemia, oxidative stress, formation of AGEs, and induction of NF-kB to the etiology of late diabetic complications. Increased glucose concentration has been shown to induce NF-kB activation in endothelial cells (38) and to increase NF-kB binding activity in peripheral blood mononuclear cells isolated from diabetic patients with poor glycemic control (37), suggesting that NF-kB activation is an early event in response to elevations in glucose contributing to diabetes-induced endothelial cell injury.
AGEs interacting with endothelial cell RAGE have been identified as relevant mediators of NF-kB activation by generating intracellular oxidative stress (39,41,76,80). Recently, it has been shown that the binding of AGEs or amyloid-P peptides to RAGE leads to perpetuated NF-kB activation in vitro and in vivo resulting in a 1-week translocation of NF-kB (p50/p65) from the cytoplasm into the nucleus (39). The AGE-RAGE-mediated NF-kB activation was initiated by the degradation of both IkBoc and IkBP. The key event in maintaining the activation of NF-kB is the induction of de novo synthesis of p65-mRNA, leading to a constantly growing pool of free NF-KBp65. Thus, AGEs are capable of activating NF-kB in vitro and in vivo, pointing to a central role of AGE-mediated NF-kB activation in late diabetic complications.
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