Managing chronic inflammation in the aging diabetic patient with CKD by diet or sevelamer carbonate: a modern paradigm shift.

The maintenance of normal metabolism and body defenses depends on the balance between cellular antioxidant and anti-inflammatory factors. This balance can be disrupted by agents/mechanisms in the extracellular milieu that induce excess reactive oxygen species (ROS) and inflammation. Cytopathic advanced glycation endproducts, present in ever increasing amounts in the modern diet, are one of the major environmental factors that cause excess ROS and/or inflammation at all ages and induce complications in aging, such as chronic kidney disease (CKD) and type 2 diabetes. Increased ROS and/or inflammation are present in both aging and CKD, and are associated with reduced cellular defenses against ROS and/or inflammation. Affected individuals have reduced defenses against further stress and are predisposed to organ failure, now a well-known phenomenon in aging. Thus, new methods are urgently needed to safely reduce ROS and/or inflammation in the aging type 2 diabetes patient with CKD. Studies of both normal aging and diabetic patients with kidney disease underline the fact that increased ROS and/or inflammation can be managed in these conditions by economical, safe, and effective interventions that reduce the uptake of advanced glycation endproducts by either modifying preparation of food or an oral drug. This communication reviews these data and adds new information on the efficacy of a drug, sevelamer carbonate, required to reduce ROS and/or inflammation in the aging type 2 diabetes patient complicated by CKD. If larger and longer studies confirm the hypothesis that one or both of these interventions reduce progression of CKD, it could represent a new paradigm in the management of complications in the type 2 diabetes patient with CKD.

Differential expression of receptors for advanced glycation end-products in peritoneal mesothelial cells exposed to glucose degradation products.

Autoclaving peritoneal dialysate fluid (PDF) degrades glucose into glucose degradation products (GDPs) that impair peritoneal mesothelial cell functions. While glycation processes leading to formation of advanced glycation end-products (AGE) were viewed commonly as being mediated by glucose present in the PDF, recent evidence indicates that certain GDPs are even more powerful inducers of AGE formation than glucose per se. In the present study, we examined the expression and modulation of AGE receptors on human peritoneal mesothelial cells (HPMC) cultured with GDPs, conventional PDF or PDF with low GDP content. HPMC cultured with GDPs differentially modulated AGE receptors (including RAGE, AGE-R1, AGE-R2 and AGE-R3) expression in a dose-dependent manner. At subtoxic concentrations, GDPs increased RAGE mRNA expression in HPMC. 2-furaldehyde (FurA), methylglyoxal (M-Glx) and 3,4-dideoxy-glucosone-3-Ene (3,4-DGE) increased the expression of AGE-R1 and RAGE, the receptors that are associated with toxic effects. These three GDPs up-regulated the AGE synthesis by cultured HPMC. In parallel, these GDPs also increased the expression of vascular endothelial growth factor (VEGF) in HPMC. PDF with lower GDP content exerted less cytotoxic effect than traditional heat-sterilized PDF. Both PDF preparations up-regulated the protein expression of RAGE and VEGF. However, the up-regulation of VEGF in HPMC following 24-h culture with conventional PDF was higher than values from HPMC cultured with PDF containing low GDP. We have demonstrated, for the first time, that in addition to RAGE, other AGE receptors including AGE-R1, AGE-R2 and AGE-R3 are expressed on HPMC. Different GDPs exert differential regulation on the expression of these receptors on HPMC. The interactions between GDPs and AGE receptors may bear biological relevance to the intraperitoneal homeostasis and membrane integrity.

Diabetes and advanced glycation endproducts.

Bio-reactive advanced glycation endproducts (AGE) alter the structure and function of molecules in biological systems and increase oxidative stress. These adverse effects of both exogenous and endogenously derived AGE have been implicated in the pathogenesis of diabetic complications and changes associated with ageing including atherosclerosis, renal, eye and neurological disease. Specific AGE receptors and nonreceptor mechanisms contribute to these processes but also assist in the removal and degradation of AGE. The final disposal of AGE depends on renal clearance. Promising pharmacologic strategies to prevent AGE formation, reduce AGE toxicity, and/or inactivate AGE are under investigation.

Accelerated diabetic glomerulopathy in galectin-3/AGE receptor 3 knockout mice.

Several molecules were shown to bind advanced glycation end products (AGEs) in vitro, but it is not known whether they all serve as AGE receptors and which functional role they play in vivo. We investigated the role of galectin-3, a multifunctional lectin with (anti)adhesive and growth-regulating properties, as an AGE receptor and its contribution to the development of diabetic glomerular disease, using a knockout mouse model. Galectin-3 knockout mice obtained by gene ablation and the corresponding wild-type mice were rendered diabetic with streptozotocin and killed 4 months later, together with age-matched nondiabetic controls. Despite a comparable degree of metabolic derangement, galectin-3-deficient mice developed accelerated glomerulopathy vs. the wild-type animals, as evidenced by the more pronounced increase in proteinuria, extracellular matrix gene expression, and mesangial expansion. This was associated with a more marked renal/glomerular AGE accumulation, indicating it was attributable to the lack of galectin-3 AGE receptor function. The galectin-3-deficient genotype was associated with reduced expression of receptors implicated in AGE removal (macrophage scavenger receptor A and AGE-R1) and increased expression of those mediating cell activation (RAGE and AGE-R2). These results show that the galectin-3-regulated AGE receptor pathway is operating in vivo and protects toward AGE-induced tissue injury in contrast to that through RAGE.

Presence of diabetic complications in type 1 diabetic patients correlates with low expression of mononuclear cell AGE-receptor-1 and elevated serum AGE.

BACKGROUND: Receptors for advanced glycation endproducts (AGE-R) mediate AGE turnover, but can also trigger inflammatory genes that promote diabetic tissue injury and diabetic complications (DC). High AGE levels and reduced AGE-R sites in kidneys of NOD mice prone to type 1 diabetes (T1D) and to renal disease (RD) suggested that impaired AGE-R function may contribute to RD in these mice. MATERIALS AND METHODS: In this study, after confirming reduced AGE-R1 expression in NOD mouse peritoneal macrophages, we tested for differences in AGE-R1, -R2, and -R3 gene expression in 54 human subjects by RT-PCR and Western analysis. Fresh peripheral blood mononuclear cells (PBMN) were isolated from 36 persons: 18 T1D patients with severe RD (DC); 11 age-and DM-duration matched patients without DC (n-DC); and 7 normal volunteers (NL). EBV-transformed lymphoblasts were obtained from an additional 18 subjects (12 T1D patients, 6 with and 6 without DC, and 6 nondiabetics). RESULTS: AGE-R1 mRNA and protein of PBMN from n-DC patients were enhanced (p < .05 versus NL) in proportion to serum AGE levels (sAGE) (p < .005 versus NL). In contrast, PBMN from DC patients exhibited no up-regulation of AGE-R1 mRNA or protein, despite higher sAGE levels (p < .005 versus NL). A similar unresponsiveness in AGE-R1 gene expression was observed in EBV-transformed lymphoblasts from DC patients versus NL (p < .01), but not in n-DC (p = NS). AGE-R2 and -R3 mRNA and protein levels were enhanced in both T1D groups (DC > n-DC) (n-DC AGE-R3, p < .05, DC AGE-R3, p < .05) compared to NL. AGE-R2 mRNA levels correlated with sAGE levels (r = .61, p < .05), and with creatinine clearance (r = -.63, p < .05). No differences were noted in AGE-R2 and -R3 mRNA expression in cultured cells. CONCLUSIONS: The consistent pattern of elevated serum AGE and low expression of AGE-R1 gene in macrophages from T1D mice (NOD), fresh PBMN and EBV-transformed cells from T1D patients with advanced DC suggests ineffective regulation of R1-mediated AGE turnover, possibly of genetic basis.

Polymorphism screening of four genes encoding advanced glycation end-product putative receptors. Association study with nephropathy in type 1 diabetic patients.

Advanced glycation end-products (AGEs) may play an important role in the pathogenesis and progression of cardiovascular and renal complications of diabetes. Four putative AGE receptors (RAGEs), AGE-R1, AGE-R2, and AGE-R3 have been described. In this study, we scanned the sequence of the genes encoding these AGE receptors in 48 patients with type 1 diabetes and investigated the identified polymorphisms (n = 19) in 199 type 1 diabetic patients with nephropathy and 193 type 1 diabetic patients without nephropathy. Overall, none of the polymorphisms was strongly associated with nephropathy. The minor allele of a polymorphism located in the promoter region of the RAGE gene (C-1152A) conferred a weak protective effect (P < 0.05) and was associated with a longer duration of nephropathy-free diabetes (P = 0.08).

Lysozyme enhances renal excretion of advanced glycation endproducts in vivo and suppresses adverse age-mediated cellular effects in vitro: a potential AGE sequestration therapy for diabetic nephropathy?

BACKGROUND: Lysozyme (LZ), a host-defense protein, contains an 18 amino-acid domain with high affinity binding for sugar-derived proteins or lipids, called advanced glycation endproducts (AGE), that are implicated in diabetes- and age-dependent complications (DC). MATERIALS AND METHODS: A) The effects of LZ on AGE- removal were tested in vivo. LZ was injected (200 ug/day, i.p., X2 weeks) in non-obese diabetic (NOD), db/db (+/+) mice, and non-diabetic, AGE-infused Sprague-Dawley rats. B) LZ: AGE interactions with macrophage-like T1B-183 cells (Mf) and mesangial cells (MC) were tested in vitro. RESULTS: A) In NOD mice, LZ reduced the elevated basal serum AGE (sAGE) (p < 0.05), enhanced urinary AGE (uAGE) excretion by approximately 2-fold (p < 0.01), while it reduced albuminuria (UA), p < 0.005. In db/db mice, LZ infusion also reduced the elevated sAGE (p < 0.05), doubled uAGE excretion (p < 0.05), and decreased UA (p < 0.01). In addition, LZ maintained normal sAGE in normal rats infused with AGE-BSA, as it doubled the urinary AGE (uAGE) clearance (p < 0.01). B) LZ stimulated the uptake and degradation of (125) I-labeled AGE-BSA and (25) I-human serum AGE by Mf, while suppressing AGE-induced TNFalpha and IGF-I production. In MC, LZ suppressed the AGE-promoted PDGF-B, alpha1 type IV collagen, and tenascin mRNA levels, and restored the AGE-suppressed expression and activity of MMP-9, but not MMP-2. CONCLUSION: LZ may act to: a) accelerate renal in-vivo AGE clearance, b) suppress macrophage and mesangial cell- specific gene activation in vitro, and c) improve albuminuria due to diabetes. These data suggest that LZ by sequestering AGEs may protect against diabetic renal damage.

The AGE-receptor in the pathogenesis of diabetic complications.

Native glucose-derived glycation derivatives (advanced glycation end products, AGE) in vascular, renal and neuronal tissues contribute to organ damage. Glycation derivatives include a number of chemically and cell-reactive substances, also termed glycoxidation products or glycotoxins (GT). Cell-associated AGE-specific receptors (AGE-Rs), AGE-R1-3, RAGE, as well as the scavenger receptors ScR-II and CD-36 that are present on vascular, renal, hemopoietic, and neuronal/glial cells, serve in the regulation of AGE uptake and removal. AGE-Rs also modulate cell activation, growth-related mediators, and cell proliferation, consequently influencing organ structure/function. This occurs via oxidant stress triggered via receptor-dependent or -independent pathways, and leads to signal activation pathways, resulting in pro-inflammatory responses. In susceptible individuals, the AGE-R expression/function may be subject to environmental or gene-related modulation, which in turn may influence tissue-specific gene functions. In this context, altered expression and activity of AGE-R components has recently been found in both mouse diabetes models and humans with diabetic complications. Although several gene polymorphisms are detected in most AGE-R components, no significant correlation to diabetic complications has as yet been found. Further investigation is underway to define whether primary or secondary genetic links of pathogenic significance exist in this system. Various AGE-binding peptides or soluble receptors have emerged as potential sequestering agents for toxic AGEs as potential therapies for diabetic complications.

Intervening in atherogenesis: lessons from diabetes.

Age-related atherogenesis resembles diabetes, a model in which glucose looms as an important culprit. Within the body, the sugar becomes a glue able to harden and obstruct blood vessels while also activating such cells as macrophages and T lymphocytes. A new discovery is that large quantities of glycation endproducts may arrive preformed in foods prepared by routine cooking methods.

Advanced glycation end-product receptor interactions on microvascular cells occur within caveolin-rich membrane domains.

Advanced glycation end products (AGEs) have an important role in diabetic complications, with many responses mediated through AGE-receptors. The current study has investigated the binding and uptake of AGEs by retinal microvascular endothelium in an attempt to understand the nature of AGE-interaction with receptors on the cell surface. There has been special emphasis placed on the R1, R2, and R3 components of AGE-receptor complex (AGE-RC) and their localization to caveolin-rich membrane domains. Retinal microvascular endothelial cells (RMECs) were exposed to either AGE-modified BSA (AGE-BSA) or native BSA conjugated to colloidal gold (gAGE, gBSA) for various time periods, fixed, and processed for transmission electron microscopy (TEM). Localization of AGE-RC components in caveolae was investigated using confocal microscopy and ultrastructural immunogold labeling. Caveolae were extracted from RMECs using differential Triton X-100 solubility, and Western analysis was conducted to test for caveolae enrichment and the presence of AGE-RC complex components. Ligand blots determined 125I-AGE-BSA binding to caveolae-enriched extracts. Colloidal gold conjugates of AGE-BSA bound to caveolae and were internalized to be trafficked to lysosomal-like compartments. AGE-receptor complex components were significantly enriched within caveolae. The data suggest that AGEs interact with their receptors within caveolae. It is significant that the AGE-R complex localizes to these organelles, because this may have implications for AGE binding, internalization, signal transduction, and the modulation of AGE-receptor-mediated vascular cell dysfunction.

Differential expression of renal AGE-receptor genes in NOD mice: possible role in nonobese diabetic renal disease.

BACKGROUND: Nonobese diabetic mice (NOD) are prone to glomerular pathology, which is accelerated with the onset of diabetes. Advanced glycation end product (AGE) interactions with AGE-receptors (AGE-Rs) in kidneys can contribute to glomerular injury and diabetic nephropathy (DN). The significant elevation in kidney AGE deposits noted in prediabetic NOD mice suggested that delayed AGE turnover in this model may contribute to its propensity toward DN. METHODS: To explore whether excess tissue AGE was linked to altered AGE-R status in the kidney, mRNA/protein expression, and of several AGE-Rs [AGE-R1, AGE-R2, AGE-R3, scavenger receptor II (ScR-II), and receptor for AGE (RAGE)], was determined in renal cortex and in mesangial cells (MCs) isolated from ND-, D-NOD, and ILE mice (N = 20 per group). Ligand binding, receptor site number, and affinity were determined in MCs from the same mouse groups. RESULTS: Prediabetic NOD kidney AGE-R1 mRNA and protein level were threefold lower than that of ILE mice (P < 0.01), while AGE-R3 mRNA was enhanced by twofold (P < 0.05) and AGE-R2, RAGE, and ScR-II mRNA remained close to normal (ILE). The onset of diabetes in NOD mice, while enhancing AGE-R1 mRNA expression by approximately twofold, failed to raise it above the normal (ILE) level, despite increases in tissue, and serum AGE. The latter was associated with higher elevation in AGE-R3 (sixfold, P < 0.05), RAGE (twofold, P = NS), and ScR-II mRNA (2. 8-fold, P = NS) above control. MCs from prediabetic NOD mice showed a threefold lower level of AGE-R1 mRNA (P < 0.02 vs. ILE) and AGE-R1-protein, and AGE-binding activity (<40% of control ILE). In contrast, AGE-R3 mRNA was enhanced (twofold), while AGE-R2 showed no change. Cultured ND-NOD MCs displayed only one fourth of the AGE-binding sites/cell present on ILE MCs (1.6 x 10(6) vs. 6.6 x 10(6), P < 0.05), which after the onset of diabetes rose to the normal range (7.0 x 10(6)/cell), but failed to exceed it. CONCLUSIONS: Reduced AGE-R1 gene expression in this strain may contribute to delayed AGE removal from and early AGE deposition in kidney tissues. This may act as a trigger for those AGE-R genes involved in growth-promoting changes, leading to DN in this strain.

A receptor for advanced glycosylation endproducts (AGEs) is colocalized with neurofilament-bound AGEs and SOD1 in motoneurons of ALS: immunohistochemical study.

Neurofilament (NF)-bound AGEs colocalize immunochemically with SOD1 in the motoneurons of patients with ALS. Among three types of AGE receptors reported in the human brain, AGE-R1 (oligosaccharyltransferase family) and AGE-R2 (substrate of protein kinase C) have been found in neurons, while AGE-R3 is restricted to glia. The present study investigates which of these receptors may be responsible for binding AGEs in the NF conglomerates of motoneurons. Immunostaining of paraffin sections from eight ALS patients (five sporadic and three familial) and three control cases was performed with antibodies directed against R1 and R2, in parallel with those against AGEs and SOD1. The sites of AGE-R1 immunoreactivity (IR) in motoneurons were in conformity to those of NF-associated AGE and SOD1 IRs. By contrast, the IR of R2 was negative in NF conglomerates. Negative R2 IR for NF conglomerates was outlined by surrounding coarse R2 immunopositive granules in the perikaryon. No IR for R1 or R2 was found in hyaline or Bunina inclusions. There was no extraneuronal expression of IR for AGE-R1 or AGEs in microglia or astroglia around the NF accumulation. The colocalization of AGE, AGE-R1, and SOD1 at NF conglomerates in motoneurons supports the notion that AGE-mediated oxidative stress and protein aggregation may be implicated in NF conglomeration and ALS pathogenesis.

Dietary glycotoxins: inhibition of reactive products by aminoguanidine facilitates renal clearance and reduces tissue sequestration.

Evidence indicates that the metabolic turnover of food-derived reactive orally absorbed advanced glycation end products (AGEs) or glycotoxins (GTs) is delayed, possibly contributing to the tissue damage induced by endogenous AGEs, especially in patients with diabetes and kidney disease. The aim of this study was to explore whether pharmacologic inhibition of dietary AGE bioreactivity by aminoguanidine (AG) can improve turnover and renal excretion of these substances. Normal Sprague-Dawley rats were fed single-labeled [14C]AGE-ovalbumin, double-labeled [14C-125I]AGE-ovalbumin, or control 125I-labeled ovalbumin diet plus free [14C]glucose, with or without AG (0.2% in water). [14C]AGE- and 125I-labeled peptide-associated radioactivity (RA) were compared with AGE immunoreactivity (by enzyme-linked immunosorbent assay) in tissues, serum, and 72-h urine samples. The effect of AG on dietary AGE bioreactivity was assessed by monitoring the inhibition of covalent complex formation between fibronectin (FN) peptide fragments and serum components, after a meal of labeled dietary AGE with or without AG. The radiolabeled AGE diet produced serum absorption and urinary excretion peaks kinetically distinct from those of free [14C]glucose or [125I]ovalbumin. Some 26% of the orally absorbed AGE-ovalbumin was excreted in the urine, whereas after AG treatment, urinary excre-tion of dietary AGEs increased markedly (to >50% of absorbed). More than 60% of tissue-bound RA was found covalently deposited in kidneys and liver, whereas after treatment with AG, tissue AGE deposits were reduced to <15% of the amount found in untreated AGE-fed controls. Sera enriched for dietary GTs formed covalently linked complexes with FN, a process completely inhibitable by AG cotreatment. Amelioration of dietary GT bioreactivity by AG improves renal elimination and prevents tissue deposition of food GTs. This may afford a novel and potentially protective use of AG against excessive tissue AGE toxicity in diabetic patients with renal disease.

Characterization of the advanced glycation end-product receptor complex in human vascular endothelial cells.

Advanced glycation end products (AGEs) have been implicated as causal factors in the vascular complications of diabetes and it is known that these products interact with cells through specific receptors. The AGE-receptor complex, originally described as p60 and p90, has been characterised in hemopoietic cells and the component proteins identified and designated AGE-R1, -R2 and -R3. In the current study we have characterised this receptor in human umbilical vein endothelial cells (HUVECs) and elucidated several important biological properties which may impact on AGE mediated vascular disease. 125I-AGE-BSA binding to HUVEC monolayers was determined with and without various cold competitors. The synthetic AGE, 2-(2-furoyl)-4(5)-furanyl-1H-imidazole (FFI)-BSA, failed to compete with AGE-BSA binding unlike observations already reported in hemopoietic cells. The ability of 125I-AGE-BSA to bind to separated HUVEC plasma membrane (PM) proteins was also examined and the binding at specific bands inhibited by antibodies to each component of the AGE-receptor complex. Western blotting of whole cell and PM fractions, before and after exposure to AGE-BSA, revealed that AGE-R1, -R2 and -R3 are subject to upregulation upon exposure to their ligand, a phenomenon which was also demonstrated by immunofluorescence of non-permeabilised cells. mRNA expression of each AGE-receptor component was apparent in HUVECs, with the AGE-R2 and -R3 gene expression being upregulated upon exposure to AGEs in a time-dependent manner. A phosporylation assay in combination with AGE-R2 immunoprecipitation demonstrated that this component of the receptor complex is phosphorylated by acute exposure to AGE-BSA. These results indicate the presence of a conserved AGE-receptor complex in vascular endothelium which demonstrates subtle differences to other cell-types. In response to AGE-modified molecules, this complex is subject to upregulation, while the AGE-R2 component also displays increased phosphorylation possibly leading to enhanced signal transduction.

Advanced glycation end products in vitreous: Structural and functional implications for diabetic vitreopathy.

PURPOSE: Advanced glycation end products (AGEs) form irreversible cross-links with many macromolecules and have been shown to accumulate in tissues at an accelerated rate in diabetes. In the present study, AGE formation in vitreous was examined in patients of various ages and in patients with diabetes. Ex vivo investigations were performed on bovine vitreous incubated in glucose to determine AGE formation and cross-linking of vitreous collagen. METHODS: By means of an AGE-specific enzyme-linked immunosorbent assay (ELISA), AGE formation was investigated in vitreous samples obtained after pars plana vitrectomy in patients with and without diabetes. In addition, vitreous AGEs were investigated in bovine vitreous collagen after incubation in high glucose, high glucose with aminoguanidine, or normal saline for as long as 8 weeks. AGEs and AGE cross-linking was subsequently determined by quantitative and qualitative assays. RESULTS: There was a significant correlation between AGEs and increasing age in patients without diabetes (r = 0.74). Furthermore, a comparison between age-matched diabetic and nondiabetic vitreous showed a significantly higher level of AGEs in the patients with diabetes (P < 0.005). Collagen purified from bovine vitreous incubated in 0.5 M glucose showed an increase in AGE formation when observed in dot blot analysis, immunogold labeling, and AGE ELISA. Furthermore, there was increased cross-linking of collagen in the glucose-incubated vitreous, when observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and protein separation. This cross-linking was effectively inhibited by coincubation with 10 mM aminoguanidine. CONCLUSIONS: This study suggests that AGEs may form in vitreous with increasing age. This process seems to be accelerated in the presence of diabetes and as a consequence of exposure to high glucose. Advanced glycation and AGE cross-linking of the vitreous collagen network may help to explain the vitreous abnormalities characteristic of diabetes.

Alzheimer's disease--synergistic effects of glucose deficit, oxidative stress and advanced glycation endproducts.

Many approaches have been undertaken to understand Alzheimer's disease (AD) but the heterogeneity of the etiologic factors makes it difficult to define the clinically most important factor determining the onset and progression of the disease. However, there is increasing evidence that the previously so-called "secondary factors" such as a disturbed glucose metabolism, oxidative stress and formation of "advanced glycation endproducts" (AGEs) and their interaction in a vicious cycle are also important for the onset and progression of AD. AGEs are protein modifications that contribute to the formation of the histopathological and biochemical hallmarks of AD: amyloid plaques, neurofibrillary tangles and activated microglia. Oxidative modifications are formed by a complex cascade of dehydration, oxidation and cyclisation reactions, subsequent to a non-enzymatic reaction of sugars with amino groups of proteins. Accumulation of AGE-crosslinked proteins throughout life is a general phenomenon of ageing. However, AGEs are more than just markers of ageing since they can also exert adverse biologic effects on tissues and cells, including the activation of intracellular signal transduction pathways, leading to the upregulation of cytokine and free radical production (oxidative stress). Oxidative stress is involved in various divergent events leading to cell damage, including an increase in membrane rigidity, DNA strand breaks and an impairment in glucose uptake. In addition, other age-related metabolic changes such as depletion of antioxidants or decreased energy production by a disturbed glucose metabolism diminish the ability of the cell to cope with the effects of radical-induced membrane, protein and DNA damage. With our improving understanding of the molecular basis for the clinical symptoms of dementia, it is hoped that the elucidation of the etiologic causes, particularly the positive feedback loops involving radical damage and a reduced glucose metabolism, will help to develop novel "neuroprotective" treatment strategies able to interrupt this vicious cycle of oxidative stress and energy shortage in AD.

Receptors for advanced glycosylation endproducts in human brain: role in brain homeostasis.

BACKGROUND: Advanced glycation end products (AGEs) are the reactive derivatives of nonenzymatic glucose-macromolecule condensation products. Aging human tissues accumulate AGEs in an age-dependent manner and contribute to age-related functional changes in vital organs. We have shown previously that AGE scavenger receptors are present on monocyte/macrophages, lymphocytes, and other cells. However, it remains unclear whether the human brain can efficiently eliminate AGE-modified proteins and whether excessive AGEs can contribute to inflammatory changes leading to brain injury in aging. MATERIALS AND METHODS: To explore the expression and characteristics of AGE-binding proteins on CNS glia components and their putative function, such as degradation of AGE-modified proteins, primary human astrocytes and human monocytes (as a microglial cell surrogate) and murine microglia (N9) cells and cell membrane extracts were used. Immunohistochemistry was used to examine the distribution of AGE-binding proteins in the human hippocampus; RT-PCR techniques were used to examine the biologic effects of AGEs and a model AGE compound, FFI, on AGE-binding protein modulation and cytokine responses of human astrocytes and monocytes. RESULTS: Our results showed that AGE-binding proteins AGE-R1, -R2, and -R3 are present in glial cells. Western blot analyses and radiolabeled ligand binding studies show that AGE-R1 and -R3 from human astrocytes bind AGE-modified proteins; binding could be blocked by anti-AGE-R1 and anti-AGE-R3 antibodies, respectively. Immunohistochemistry showed that AGE-R1 and -R2 are expressed mainly in neurons; only some glial cells express these AGE-binding proteins. In contrast, AGE-R3 was found only on those astrocytes whose positively stained foot processes extend and surround the sheath of microcapillaries. RT-PCR results showed that mRNAs of the three AGE-binding proteins are expressed constitutively in human astrocytes and monocytes, and receptor transcripts are not regulated by exogenous AGEs, the model AGE compound FFI, or phorbol ester. At the concentrations used, GM-CSF appears to be the only cytokine whose transcript and protein levels are regulated in human astrocytes by exogenous AGEs. CONCLUSIONS: The selective presence of AGE-binding proteins in pyramidal neurons and glial cells and their roles in degrading AGE-modified protein in glial cells suggest that the human brain has a mechanism(s) to clear AGE-modified proteins. Without this capacity, accumulation of AGEs extracellularly could stimulate glial cells to produce the major inflammatory cytokine GM-CSF, which has been shown to be capable of up-regulating AGE-R3. It remains to be determined whether AGE-binding proteins could be aberrant or down-regulated under certain pathological conditions, resulting in an insidious inflammatory state of the CNS in some aging humans.

Elevated AGE-modified ApoB in sera of euglycemic, normolipidemic patients with atherosclerosis: relationship to tissue AGEs.

BACKGROUND: Advanced glycation endproducts (AGEs) are implicated in the pathogenesis of atherosclerotic vascular disease of diabetic and nondiabetic etiology. Recent research suggests that advanced glycation of ApoB contributes to the development of hyperlipidemia. AGE-specific receptors, expressed on vascular endothelium and mononuclear cells, may be involved in both the clearance of, and the inflammatory responses to AGEs. The aim of this study was to examine whether there is a relationship between serum AGE-ApoB and AGEs in arterial tissue of older normolipidemic nondiabetic patients with occlusive atherosclerotic disease, compared with age-matched and younger asymptomatic persons. MATERIALS AND METHODS: Serum AGE-ApoB was measured by ELISA in 21 cardiac bypass patients. Furthermore, an AGE-specific monoclonal antibody, and polyclonal antibodies against anti-AGE-receptor (anti-AGE-R) 1 and 2 were used to explore the localization and distribution of AGEs and AGE-R immunoreactivity (IR) in arterial segments excised from these patients. RESULTS: Serum AGE-ApoB levels were significantly elevated in the asymptomatic, older population, compared with those in young healthy persons (259 +/- 24 versus 180 +/- 21 AGE U/mg of ApoB, p < 0.01). Higher AGE-ApoB levels were observed in those patients with atherosclerosis (329 +/- 23 versus 259 +/- 24 AGE U/mg ApoB, p < 0.05). Comparisons of tissue AGE-collagen with serum AGE-ApoB levels showed a significant correlation (r = 0.707, p < 0.01). In early lesions, AGE-IR occurred mostly extracellularly. In fatty streaks and dense, cellular atheromatous lesions, AGE-IR was visible within lipid-containing smooth muscle cells and macrophages, while in late-stage, acellular plaques, AGE-IR occurred mostly extracellularly. AGE-R1 and -R2 were observed on vascular endothelial and smooth-muscle cells and on infiltrating mononuclear cells in the early-stage lesions, whereas in dense, late-stage plaques, they colocalized mostly with lipid-laden macrophages. On tissue sections, scoring of AGE-immunofluorescence correlated with tissue AGE and plasma AGE-ApoB. CONCLUSIONS: (1) The correlation between arterial tissue AGEs and circulating AGE-ApoB suggests a causal link between AGE modification of lipoproteins and atherosclerosis. AGE-specific receptors may contribute to this process. (2) Serum AGE-ApoB may serve to predict atherosclerosis in asymptomatic patients.

Upregulation of mesangial growth factor and extracellular matrix synthesis by advanced glycation end products via a receptor-mediated mechanism.

Enhanced advanced glycosylation end product (AGE) formation has been shown to participate in the pathogenesis of diabetes-induced glomerular injury by mediating the increased extracellular matrix (ECM) deposition and altered cell growth and turnover leading to mesangial expansion. These effects could be exerted via an AGE-receptor-mediated upregulation of growth factors, such as the IGFs and transforming growth factor-beta (TGF-beta). We tested this hypothesis in human and rat mesangial cells grown on nonglycated or native bovine serum albumin (BSA), glycated BSA with AGE formation (BSA-AGE), or glycated BSA in which AGE formation was prevented by the use of aminoguanidine (BSA-AM), in the presence or absence of an antibody, alpha-p60, directed against the p60/OST protein named AGE-receptor 1 (AGE-R1), or normal control (pre-immune) serum. The mRNA and/or protein levels of IGF-I, IGF-II, IGF receptors, IGF binding proteins (IGFBPs), TGF-beta1 and the ECM components fibronectin, laminin, and collagen IV were measured, together with cell proliferation. Both human and rat mesangial cells grown on BSA-AGE showed increased IGF-I and total and bioactive TGF-beta medium levels and enhanced IGF-I, IGF-II, and TGF-beta1 gene expression, compared with cells grown on BSA, whereas total IGFBP and IGFBP-3 medium content, IGF receptor density and affinity, and IGF-I receptor transcripts were unchanged. Moreover, cells grown on BSA-AGE showed increased ECM protein and mRNA levels versus cells cultured on BSA, whereas cell proliferation was unchanged in human mesangial cells and slightly reduced in rat mesangial cells. Growing cells on BSA-AM did not affect any of the measured parameters. Co-incubation of BSA-AGE with anti-AGE-R1, but not with pre-immune serum, prevented AGE-induced increases in IGF-I, TGF-beta1, and ECM production or gene expression; anti-AGE-R1 also reduced growth factor and matrix synthesis in cells grown on BSA. These results demonstrate that mesangial IGF and TGF-beta1 synthesis is upregulated by AGE-modified proteins through an AGE-receptor-mediated mechanism. The parallelism with increased ECM production raises the speculation that the enhanced synthesis of these growth factors resulting from advanced nonenzymatic glycation participates in the pathogenesis of hyperglycemia-induced mesangial expansion.