The elevated glycation of macromolecules by the reactive dicarbonyl and α-oxoaldehyde methylglyoxal (MG) has been associated with diabetes and its complications. We have identified a rare flavone, fisetin, which increases the level and activity of glyoxalase 1, the enzyme required for the removal of MG, as well as the synthesis of its essential co-factor, glutathione. It is shown that fisetin reduces two major complications of diabetes in Akita mice, a model of type 1 diabetes. Although fisetin had no effect on the elevation of blood sugar, it reduced kidney hypertrophy and albuminuria and maintained normal levels of locomotion in the open field test. This correlated with a reduction in proteins glycated by MG in the blood, kidney and brain of fisetin-treated animals along with an increase in glyoxalase 1 enzyme activity and an elevation in the expression of the rate-limiting enzyme for the synthesis of glutathione, a co-factor for glyoxalase 1. The expression of the receptor for advanced glycation end products (RAGE), serum amyloid A and serum C-reactive protein, markers of protein oxidation, glycation and inflammation, were also increased in diabetic Akita mice and reduced by fisetin. It is concluded that fisetin lowers the elevation of MG-protein glycation that is associated with diabetes and ameliorates multiple complications of the disease. Therefore, fisetin or a synthetic derivative may have potential therapeutic use for the treatment of diabetic complications.

OBJECTIVE

Advanced glycation end products (AGEs) and endothelial progenitor cells (EPCs) play key roles in pathogenesis of diabetes-related vascular complications. AGEs can induce dysfunction in EPCs. The peroxisome proliferator-activated receptor-gamma (PPARgamma) agonists are widely used in the treatment of type 2 diabetes, and it remains unknown if they could attenuate EPC dysfunction induced by AGEs.

METHODS

EPCs isolated from healthy adults were cultured with various concentrations of AGEs (0, 50, 100 and 200 mg L(-1)) with or without rosiglitazone (10 nM), antibody for the receptors for AGE-human serum albumin (anti-receptor for advanced glycation end products (RAGE); 50 microg mL(-1)), phosphatidylinositol-3-kinase (PI3K) inhibitor (LY294002, 5 microM), nitric oxide (NO) synthase inhibitor (L-N(G)-nitro-arginine methyl ester (L-NAME), 100 microM) or sodium nitroprusside (SNP, 25 microM). Proliferation, apoptosis, cell adhesion, migration and NO production in EPCs were assessed, and expressions of endothelial NO synthase (eNOS) and Akt were determined.

RESULTS

Number, proliferation/migration capacities, eNOS and Akt phosphorylation as well as NO synthesized by EPCs were increased by rosiglitazone and reduced by AGEs. AGEs promoted while rosiglitazone reduced EPC apoptosis. The AGE-induced effects were significantly ameliorated by pre-incubation with rosiglitazone, RAGE antibody and SNP. The beneficial effects of rosiglitazone could be blocked by pretreatment with L-NAME and LY294002.

CONCLUSIONS

The PPARgamma agonist rosiglitazone increased EPC function and attenuated EPC dysfunction induced by AGEs via upregulating the Akt-eNOS signal pathways of EPCs.

Inhibitor of NF-kappaB kinases beta (IKKbeta) and alpha (IKKalpha) activate distinct NF-kappaB signaling modules. The IKKbeta/canonical NF-kappaB pathway rapidly responds to stress-like conditions, whereas the IKKalpha/noncanonical pathway controls adaptive immunity. Moreover, IKKalpha can attenuate IKKbeta-initiated inflammatory responses. High mobility group box 1 (HMGB1), a chromatin protein, is an extracellular signal of tissue damage-attracting cells in inflammation, tissue regeneration, and scar formation. We show that IKKalpha and IKKbeta are each critically important for HMGB1-elicited chemotaxis of fibroblasts, macrophages, and neutrophils in vitro and neutrophils in vivo. By time-lapse microscopy we dissected different parameters of the HMGB1 migration response and found that IKKalpha and IKKbeta are each essential to polarize cells toward HMGB1 and that each kinase also differentially affects cellular velocity in a time-dependent manner. In addition, HMGB1 modestly induces noncanonical IKKalpha-dependent p52 nuclear translocation and p52/RelB target gene expression. Akin to IKKalpha and IKKbeta, p52 and RelB are also required for HMGB1 chemotaxis, and p52 is essential for cellular orientation toward an HMGB1 gradient. RAGE, a ubiquitously expressed HMGB1 receptor, is required for HMGB1 chemotaxis. Moreover, IKKbeta, but not IKKalpha, is required for HMGB1 to induce RAGE mRNA, suggesting that RAGE is at least one IKKbeta target involved in HMGB1 migration responses, and in accord with these results enforced RAGE expression rescues the HMGB1 migration defect of IKKbeta, but not IKKalpha, null cells. Thus, proinflammatory HMGB1 chemotactic responses mechanistically require the differential collaboration of both IKK-dependent NF-kappaB signaling pathways.

Epigenetic editing of gene expression by aberrant methylation of DNA may help tumor cells escape attack from the innate and acquired immune systems. Resistance to antiproliferative effects and apoptosis induction by interferons (IFNs) was postulated to result from silencing of IFN response genes by promoter hypermethylation. Treatment of human ACHN renal cell carcinoma (RCC) and A375 melanoma cells with the DNA demethylating nucleoside analog 5-AZA-2'-deoxycytidine (5-AZA-dC) synergistically augmented antiproliferative effects of IFN- alpha (alpha) 2 and IFN-beta (beta). Either 5-AZA-dC or an antisense to DNA methyltransferase 1 (DNMT1) overcame resistance to apoptosis induction by IFNs with up to 85% apoptotic cells resulting from the combinations. No similar potentiation occurred in normal kidney epithelial cells. IFN response genes were augmented more than 10 times in expression by 5-AZA-dC. Demethylation by 5-AZA-dC of the promoter of the prototypic, apoptosis-associated IFN response gene XAF1 was confirmed by methylation-specific polymerase chain reaction. siRNA to XAF1 inhibited IFN-induced apoptosis; conversely, overexpression of XAF1 overcame resistance to apoptosis induction by IFN-beta. As occurred with apoptosis-resistant melanoma cells in vitro, tumor growth inhibition in the nude mouse of human A375 melanoma xenografts resulted from treatment with 5-AZA-dC in combination with IFN-beta, an effect not resulting from either single agent. The importance of epigenetic remodeling of expression of immune-modifying genes in tumor cells was further suggested by identifying reactivation of the cancer-testis antigens MAGE and RAGE in ACHN cells after DNMT1 depletion. Thus, inhibitors of DNMT1 may have clinical relevance for immune modulation by augmentation of cytokine effects and/or expression of tumor-associated antigens.

The mechanisms underlying diabetes-mediated bone loss are not well defined. It has been reported that the advanced glycation endproducts (AGEs) and receptor for AGEs (RAGEs) are involved in diabetic complications. Here, mice deficient in RAGE were used as a model for investigating the effects of RAGE on bone mass. We found that RAGE-/- mice have a significantly increased bone mass and bone biomechanical strength and a decreased number of osteoclasts compared to wild-type mice. The serum levels of IL-6 and bone breakdown marker pyridinoline were significantly decreased in RAGE-/- mice. RAGE-/- mice maintain bone mass following ovariectomy, whereas wild-type mice lose bone mass. Furthermore, osteoclast-like cells do express RAGE mRNA. Our data therefore indicate that RAGE serves as a positive factor to regulate the osteoclast formation, directly implicates a role for RAGE in diabetes-promoted bone destruction, and documents that the AGE-RAGE interaction may account for diabetes associated bone loss.

Advanced glycation end products (AGEs), a heterogeneous group of compounds formed by nonenzymatic glycation reactions between reducing sugars and amino acids, lipids, or DNA, are formed not only in the presence of hyperglycemia, but also in diseases associated with high levels of oxidative stress, such as CKD. In chronic renal failure, higher circulating AGE levels result from increased formation and decreased renal clearance. Interactions between AGEs and their receptors, including advanced glycation end product-specific receptor (RAGE), trigger various intracellular events, such as oxidative stress and inflammation, leading to cardiovascular complications. Although patients with CKD have a higher burden of cardiovascular disease, the relationship between AGEs and cardiovascular disease in patients with CKD is not fully characterized. In this paper, we review the various deleterious effects of AGEs in CKD that lead to cardiovascular complications and the role of these AGEs in diabetic nephropathy. We also discuss potential pharmacologic approaches to circumvent these deleterious effects by reducing exogenous and endogenous sources of AGEs, increasing the breakdown of existing AGEs, or inhibiting AGE-induced inflammation. Finally, we speculate on preventive and therapeutic strategies that focus on the AGE-RAGE axis to prevent vascular complications in patients with CKD.

The multiligand receptor for advanced glycation end products (RAGE) of the immunoglobulin superfamily is expressed on multiple cell types implicated in the immune-inflammatory response and in atherosclerosis. Multiple studies have elucidated that ligand-RAGE interaction on cells, such as monocytes, macrophages, and endothelial cells, mediates cellular migration and upregulation of proinflammatory and prothrombotic molecules. In addition, recent studies reveal definitive rules for RAGE in effective T lymphocyte priming in vivo. RAGE ligand AGEs may be formed in diverse settings; although AGEs are especially generated in hyperglycemia, their production in settings characterized by oxidative stress and inflammation suggests that these species, in part via RAGE, may contribute to the pathogenesis of atherosclerosis. In murine models of atherosclerosis, vascular inflammation is a key factor and one which is augmented, in parallel with even further increases in RAGE ligands, in diabetic macrovessels. The findings that antagonism and genetic disruption of RAGE in atherosclerosis-susceptible mice strikingly reduces vascular inflammation and atherosclerotic lesion area and complexity link RAGE intimately to these processes and suggest that RAGE is a logical target for therapeutic intervention in aberrant inflammatory mechanisms and in atherosclerosis.

Familial amyloid polyneuropathy (FAP) is a neurodegenerative disorder associated with extracellular deposition of mutant transthyretin (TTR) amyloid fibrils, particularly in the peripheral nervous system. We have hypothesized that binding of TTR fibrils to the receptor for advanced glycation end products (RAGE) on critical cellular targets is associated with a destructive stress response underlying peripheral nerve dysfunction. Analysis of nerve biopsy samples from patients with FAP (n = 16) at different stages of disease (0-3), compared with age-matched controls (n = 4), by semiquantitative immunohistology and in situ hybridization showed increased levels of RAGE, beginning at the earliest stages of the disease (FAP 0; p < 0.02) and especially localized in axons. Upregulation of proinflammatory cytokines (tumor necrosis factor-alpha and interleukin-1beta) (approximately threefold; p < 0.02) and the inducible form of nitric oxide synthase (iNOS) ( approximately 2.5-fold; p < 0.04) was also observed in a distribution overlapping RAGE expression. Tyrosine nitration and increased activated caspase-3 in axons from FAP patients (p < 0.03) were apparent. Although these data suggest the presence of ongoing neuronal stress, there was no upregulation of neurotrophins (nerve growth factor and neurotrophin-3) in FAP nerves. Studies on cultured neuronal-like, Schwann, and endothelial cells incubated with TTR fibrils displayed RAGE-dependent expression of cytokines and iNOS at early times (6 and 12 hr, respectively), followed by later (24 hr) activation of caspase-3 and DNA fragmentation. We propose that the interaction of TTR fibrils with RAGE may contribute to cellular stress and toxicity in FAP. Furthermore, there is an apparent lack of responsiveness of Schwann cells in FAP nerve to provide neurotrophic factors.

S100A12, also called EN-RAGE (extracellular newly identified receptor for advanced glycation end products binding protein) or calcium-binding protein in amniotic fluid-1, is a ligand for RAGE. It has been shown that S100A12 induces adhesion molecules such as vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 in the vascular endothelial cell and mediates migration and activation of monocytes/macrophages through RAGE binding and that infusion of lipopolysaccharide into mice causes time-dependent increase of S100A12 in the plasma. Therefore, circulating S100A12 protein may be involved in chronic inflammation in the atherosclerotic lesion. In this study, we developed an ELISA system that uses specific monoclonal antibodies against recombinant human S100A12 to measure plasma S100A12 levels in patients with diabetes. On using our S100A12 ELISA system, the coefficients of variation of intra- and interassay were less than 4 and 9%, respectively. The analytical lower detection limit was 0.2 ng/ml. When plasma S100A12 levels were measured by this system, the concentrations were more than twice as high in the patients with diabetes, compared with those without. Using univariate analysis in all subjects, plasma S100A12 concentrations correlated with hemoglobin A1c, fasting glucose, high-sensitivity C-reactive protein and white blood cell count. Stepwise multiple regression analyses, however, revealed that only white blood cell count and hemoglobin A1c remained significant independent determinants of plasma S100A12 concentration. These results suggest that plasma S100A12 protein levels are regulated by factors related to subclinical inflammation and glucose control in patients with type 2 diabetes.

Melatonin acts as a pleiotropic agent in various age-related neurodegenerative diseases. In this study, we examined the underlying neuroprotective mechanism of melatonin against D-galactose-induced memory and synaptic dysfunction, elevated reactive oxygen species (ROS), neuroinflammation and neurodegeneration. D-galactose was administered (100 mg/kg intraperitoneally (i.p.)) for 60 days. After 30 days of D-galactose administration, vehicle (same volume) or melatonin (10 mg/kg, i.p.) was administered for 30 days. Our behavioral (Morris water maze and Y-maze test) results revealed that chronic melatonin treatment alleviated D-galactose-induced memory impairment. Additionally, melatonin treatment reversed D-galactose-induced synaptic disorder via increasing the level of memory-related pre-and postsynaptic protein markers. We also determined that melatonin enhances memory function in the D-galactose-treated mice possibly via reduction of elevated ROS and receptor for advanced glycation end products (RAGE). Furthermore, Western blot and morphological results showed that melatonin treatment significantly reduced D-galactose-induced neuroinflammation through inhibition of microgliosis (Iba-1) and astrocytosis (GFAP), and downregulating other inflammatory mediators such as p-IKKβ, p-NF-K B65, COX2, NOS2, IL-1β, and TNFα. Moreover, melatonin lowered the oxidative stress kinase p-JNK which suppressed various apoptotic markers, that is, cytochrome C, caspase-9, caspase-3 and PARP-1, and prevent neurodegeneration. Hence, melatonin attenuated the D-galactose-induced memory impairment, neuroinflammation and neurodegeneration possibly through RAGE/NF-K B/JNK pathway. Taken together, our data suggest that melatonin could be a promising, safe and endogenous compatible antioxidant candidate for age-related neurodegenerative diseases such as Alzheimer's disease (AD).

The engagement of the receptor for advanced glycation end products (RAGE) on the cell surface induces cellular dysfunction in a number of pathophysiological situations of vascular dysfunction, tumor cell invasion, inflammatory response, and T cell infiltration. The administration of truncated, soluble RAGE can modulate RAGE-mediated perturbations. Here, we report a novel splice variant (delta8-RAGE) of RAGE mRNA, which lacks exon 8 of the genomic RAGE gene and contains an early stop codon in exon 10 due to a frame shift mutation. delta8-RAGE mRNA was found in human primary astrocytes and peripheral blood mononuclear cells (PBMCs). Transient transfection experiments demonstrated that delta8-RAGE mRNA was translated into a secretory protein as deduced. Moreover, two different segments of the spliced variant were identified in PBMCs by RT-PCR. The findings of this study suggest that the diverse splicing variants of RAGE are possible in many tissues and their products may influence the RAGE-mediated pathogenesis and immune modulation.

Advanced glycation end products play major roles in diabetic complications. They act via their receptor RAGE to induce inflammatory genes such as cyclooxygenase-2 (COX-2). We examined the molecular mechanisms by which the RAGE ligand, S100b, induces COX-2 in monocytes. S100b significantly increased COX-2 mRNA accumulation in THP-1 monocytes at 2 h via mRNA stability. This was further confirmed by showing that S100b increased stability of luciferase-COX-2 3'-UTR mRNA. Chromatin immunoprecipitation and RNA immunoprecipitation revealed that S100b decreased occupancy of the DNA/RNA-binding protein, heterogeneous nuclear ribonuclear protein K (hnRNPK), at the COX-2 promoter but simultaneously increased its binding to the COX-2 3'-UTR. S100b treatment promoted the translocation of nuclear hnRNPK to cytoplasm, whereas a cytoplasmic translocation-deficient hnRNPK mutant inhibited S100b-induced COX-2 mRNA stability. Small interfering RNA-mediated specific knockdown of hnRNPK blocked S100b-induced COX-2 mRNA stability, whereas on the other hand, overexpression of hnRNPK increased S100b-induced COX-2 mRNA stability. S100b promoted the release of entrapped COX-2 mRNA from cytoplasmic processing bodies, sites of mRNA degradation. Furthermore, S100b significantly down-regulated the expression of a key microRNA, miR-16, which can destabilize COX-2 mRNA by binding to its 3'-UTR. MiR-16 inhibitor oligonucleotides increased, whereas, conversely, miR-16 mimic oligonucleotides decreased COX-2 mRNA stability in monocytes, further supporting the inhibitory effects of miR-16. Interestingly, hnRNPK knockdown increased miR-16 binding to COX-2 3'-UTR, indicating a cross-talk between them. These new results demonstrate that diabetic stimuli can efficiently stabilize inflammatory genes via opposing actions of key RNA-binding proteins and miRs.

Advanced glycation end-products (AGEs) are formed from the so-called Amadori products by rearrangement followed by other reactions giving rise to compounds bound irreversibly. The structure of some of them is shown and the mechanism of formation is described. Several AGE binding molecules (Receptors for AGE, RAGE) are known and it is thought that many of the effects caused by AGEs are mediated by RAGE. Some of these were shown to be toxic, and called TAGE. The mechanism of detoxification of glyoxal and methylglyoxal by the glyoxalase system is described and also the possibility to eliminate glycated proteins by deglycation enzymes. Compounds able to inhibit AGEs formation are also taken into consideration.

BACKGROUND

Chronic exposure to peritoneal dialysis fluid (PDF) affects the peritoneum, but precise causative factors are incompletely understood. We examined the effects of standard and new PDF on peritoneal function and structure.

METHODS

Female Wistar rats received twice daily intraperitoneal infusions of a standard lactate-buffered 3.86% glucose PDF at pH 5.5 (Dianeal) (N= 12), a low glucose degradation product (GDP) containing bicarbonate/lactate-buffered 3.86% glucose PDF at pH 7.4 (Physioneal) (N= 12), a lactate-buffered amino acid-based PDF at pH 6.7 (Nutrineal) (N= 12) or Earle's Balanced Salt Solution at pH 7.4 (EBSS) (N= 12) during 12 weeks.

RESULTS

Net ultrafiltration was lower after treatment with standard PDF, but not with low-GDP bicarbonate/lactate-buffered and amino acid-based PDF, compared to EBSS. Peritonea exposed to standard PDF were characterized by an increased expression of vascular endothelial growth factor (VEGF), microvascular proliferation as well as submesothelial fibrosis, which were not observed in other groups. Staining for methylglyoxal adducts was prominent in the standard PDF-exposed group, mild in the low GDP bicarbonate/lactate-buffered group and absent in the other groups. Standard PDF induced accumulation of advanced glycation end products (AGEs) and up-regulation of the receptor for AGE (RAGE). AGEs accumulation was absent and RAGE expression was only modestly increased in low-GDP bicarbonate/lactate-buffered and amino acid-based PDF.

CONCLUSIONS

Long-term in vivo exposure to standard PDF adversely affects peritoneal function and structure. A low-GDP bicarbonate/lactate-buffered and amino acid-based PDF better preserved peritoneal integrity and may thus improve the longevity of the peritoneal membrane. GDPs and associated accelerated AGE formation are the main causative factors in PDF-induced peritoneal damage.

OBJECTIVE

Using primary cultures of mouse hippocampal neurons, we studied the molecular and functional interactions between high mobility group box-1 (HMGB1) and the N-methyl-d-aspartate receptor (NMDAR), two proteins playing a key role in neuronal hyperexcitability. By measuring NMDA-induced calcium (Ca(2+)) increase in neuronal somata and neurotoxicity as functional read-out parameters, we explored the role of the redox state of HMGB1, the receptor involved, and the molecular signaling underlying its interactions with postsynaptic NMDAR. We also investigated whether HMGB1 redox state affects its proconvulsive effects in mice.

RESULTS

Nonoxidizable HMGB1 with a triple cysteine-to-serine replacement (3S-HMGB1) was ineffective on NMDA response. Conversely, the disulfide-containing form of HMGB1 dose dependently enhanced NMDA-induced Ca(2+) increase in neuronal cell bodies. This effect was prevented by BoxA, a competitive HMGB1 antagonist, and by Rhodobacter sphaeroides lipopolysaccharide (LPS-RS), a toll-like receptor 4 (TLR4) selective antagonist, and it was abrogated in neurons lacking TLR4 while persisting in the absence of receptor for advanced glycation end products (RAGE). TLR4 and NMDAR subunit 1 (NR1) and 2B (NR2B) were colocalized in neurons. Disulfide HMGB1 effect on NMDA-induced Ca(2+) influx was prevented by 3-O-methylsphingomyelin (3-O-MS) and 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3,4-d] pyrimidine, (PP2) selective inhibitors of neutral sphingomyelinase and Src-family Tyr kinases, respectively. Disulfide HMGB1, but not 3S-HMGB1, increased Tyr(1472) phosphorylation of the NR2B subunit of the NMDAR, which is known to increase Ca(2+) channel permeability. Similarly, disulfide HMGB1 increased NMDA-induced neuronal cell death in vitro and enhanced kainate-induced seizures in vivo.

CONCLUSIONS

We describe a novel molecular neuronal pathway activated by HMGB1 that could be targeted in vivo to prevent neurodegeneration and seizures mediated by excessive NMDARs stimulation.

Advanced glycation end products (AGE) have been observed in various pathological conditions especially in diabetes mellitus. However, it is unclear as to whether AGE are involved in insulin resistance in adipose tissues. In this study, we examined the effects of AGE on insulin sensitivity in adipocytes by examining the effects of AGE and its mechanisms on the glucose uptake in adipocytes and adipocyte differentiation. Glucose-, glyceraldehyde-, or glycolaldehyde-derived AGE inhibited the differentiation of 3T3-L1 cells. These AGE also inhibited the glucose uptake in the absence or presence of insulin, which were completely prevented by antibody against AGE or receptor for AGE (RAGE). The AGE increased the intracellular reactive oxygen species (ROS) generation in 3T3-L1 adipocytes, and the effects of AGE on glucose uptake were completely reversed by the treatment with an anti-oxidant, N-acetylcysteine. The AGE also induced the expression of monocyte chemoattractant protein-1, which has been implicated in the development of obesity-associated glucose intolerance, in 3T3-L1 adipocytes. Our present study suggests that AGE-RAGE interaction inhibits the glucose uptake through the overgeneration of intracellular ROS, thus indicating that it is involved in the development of obesity-related insulin resistance.

The receptor for advanced glycation end products (RAGE) contributes to the inflammatory response in many acute and chronic diseases. In this context, RAGE has been identified as a ligand for the beta(2)-integrin Mac-1 under static in vitro conditions. Because intercellular adhesion molecule (ICAM)-1 also binds beta(2)-integrins, we studied RAGE(-/-), Icam1(-/-), and RAGE(-/-) Icam1(-/-) mice to define the relative contribution of each ligand for leukocyte adhesion in vivo. We show that trauma-induced leukocyte adhesion in cremaster muscle venules is strongly dependent on RAGE and ICAM-1 acting together in an overlapping fashion. Additional in vivo experiments in chimeric mice lacking endothelium-expressed RAGE and ICAM-1 located the adhesion defect to the endothelial compartment. Using microflow chambers coated with P-selectin, CXCL1, and soluble RAGE (sRAGE) demonstrated that sRAGE supports leukocyte adhesion under flow conditions in a Mac-1- but not LFA-1-dependent fashion. A static adhesion assay revealed that wild-type and RAGE(-/-) neutrophil adhesion and spreading were similar on immobilized sRAGE or fibrinogen. These observations indicate a crucial role of endothelium-expressed RAGE as Mac-1 ligand and uncover RAGE and ICAM-1 as a new set of functionally linked adhesion molecules, which closely cooperate in mediating leukocyte adhesion during the acute trauma-induced inflammatory response in vivo.

The role of chronic hyperglycemia in the development of diabetic microvascular complications and in neuropathy has been clearly established by intervention studies. However, the biochemical or cellular links between elevated blood glucose levels, and the vascular lesions remain incompletely understood. This review focuses on the consequences of hyperglycemia on the formation of advanced glycation end-products (AGEs), and on the role of AGEs and of their specific receptors (RAGE) in the functional and anatomical alterations of the vascular wall. AGEs are formed during the Maillard reaction by the binding of aldoses on free NH(2) groups of proteins, which, after a cascade of molecular rearrangements, result in molecules of brown color and specific fluorescence. Experimental studies have indicated that the binding of AGEs to RAGE activates cells, particularly monocytes and endothelial cells. Activated endothelial cells produce cytokines, and express adhesion molecules and tissue factor. The role of AGEs in increased oxidative stress, and in the functional alterations in vascular tone control observed in diabetes, in part related to a reduction in nitric oxide, is also discussed. The microvascular retinal, glomerular and nerve lesions induced by experimental diabetes in animals are prevented by an inhibitor of AGEs formation, aminoguanidine. The administration in diabetic animals of recombinant RAGE, which hinders AGEs-RAGE interaction, prevents hyperpermeability and vascular lesions. These data suggest a central role of AGEs and RAGE in the development of chronic complications of diabetes.

The receptor for advanced glycation end products (RAGE) has been proposed as a cell surface receptor that binds amyloid-beta protein (Abeta), thereby triggering its cytotoxic effects [S.D. Yan, X. Chen, J. Fu, M. Chen, H. Zhu, A. Roher, T. Slattery, L. Zhao, M. Nagashima, J. Morser, A. Migheli, P. Nawroth, D. Stern, A.M. Schmidt, RAGE and amyloid-beta peptide neurotoxicity in Alzheimer's disease, Nature 382 (1996) 685-691.]. A cDNA library of human lung was screened for RAGE with an appropriate hybridization probe. In addition to cell surface RAGE, one clone was found which encodes a new version of RAGE, termed hRAGEsec, which lacks the 19 amino acids of the membrane-spanning region and is therefore secreted. Comparison with the genomic sequence revealed that the synthesis of the secreted isoform requires alternative splicing. The deduced protein sequence of the mature hRAGEsec consists of 321 amino acids with a predicted molecular mass of 35.66 kDa. The pattern of expression of hRAGEsec in human brain was analyzed by in situ hybridization histochemistry. The most intense expression of the gene in contrast to cell surface RAGE was detected in hippocampal CA3 pyramidal cells, dentate gyrus granule cells, cortical neurons as well as glial cells in white matter. To investigate the interaction between Abeta and RAGE and another scavenger receptor, SRA, under physiological conditions, they were co-expressed with human betaAPP(695)-SFAD in a human cell and the level of Abeta in the condition medium was assessed by immunoprecipitation and enzyme-linked immunosorbent assay (ELISA) analysis. A nearly 100% reduction of Abeta from the conditioned medium of hRAGE cells and approximately 40% reduction from the SRA-cells implied that hRAGE could be a prominent cell surface receptor interacting with Abeta.

Mutated transthyretin (TTR) fibrils are associated with the pathology of familial amyloidotic polyneuropathy (FAP), in which extracellular amyloid deposits lead to degeneration of cells and tissues, in particular neurons of the peripheral nerve. Here we present evidence that the receptor for advanced glycation end products (RAGE), previously associated with Alzheimer's disease, acts as a selective cell surface acceptor site for both soluble and fibrillar TTR. Immunohistochemical studies demonstrating increased expression of RAGE in FAP tissues suggested the relevance of this receptor to TTR-induced fibrillar pathology. In vitro studies using soluble RAGE showed saturable specific interaction with soluble and fibrillar TTR with a K(d) of approximately 120 nM. However, no binding was observed when soluble TTR was combined with retinol-binding protein, which represents the form in which TTR normally circulates in plasma. Specific binding of TTR to RAGE-transfected Chinese hamster ovary cells (which was completely blocked by anti-RAGE) was observed, confirming that RAGE could mediate TTR binding to cellular surfaces. RAGE-dependent activation of nuclear transcription factor kB (NF-kB) by TTR fibrils was shown in PC-12 cells stably transfected to overexpress the receptor. Furthermore, FAP nerves showed up-regulation of p50, one of the NF-kB subunits, when compared with age-matched controls. From these observations we predict that, in vivo, the presence of TTR fibrils associated with cellular surfaces of FAP patients, by contributing to NF-kB activation, leads to the pathogenesis of neurodegeneration. Further insights into the consequences of the interaction of fibrillar TTR with RAGE may therefore provide a better understanding of neurodegeneration associated with FAP.

The accumulation of plasma advanced oxidation protein products (AOPPs) is prevalent in chronic kidney disease. We previously showed that accumulation of AOPPs resulted in podocyte apoptosis and their deletion by a cascade of signaling events coupled with intracellular oxidative stress. The transmembrane receptor that specifically transmits the AOPPs' signals to elicit cellular activity, however, remains unknown. Using co-immunoprecipitation and immunofluorescence, we found that AOPPs colocalized and interacted with the receptor of advanced glycation end products (RAGE) on podocytes. Blocking RAGE by anti-RAGE immunoglobulin G or its silencing by siRNA significantly protected podocytes from AOPPs-induced apoptosis both in vitro and in vivo and ameliorated albuminuria in AOPPs-challenged mice. AOPPs-induced activation of nicotinamide adenine dinucleotide phosphate oxidase and the excessive generation of intracellular superoxide were largely inhibited by anti-RAGE immunoglobulin G or RAGE siRNA. Moreover, blockade of RAGE decreased the activation of the p53/Bax/caspase-dependent proapoptotic pathway induced by AOPPs. Thus, AOPPs interact with RAGE to induce podocyte apoptosis and this, in part, may contribute to the progression of chronic kidney disease.

Mammalian Toll-like receptors (TLRs) are cellular pattern-recognizing receptors (PRRs) that recognize the molecular patterns of pathogens. After engaging the pathogenic patterned ligands, the cytosolic portion of the TLRs in monocytes and macrophages, recruits adaptor proteins, via a receptor-driven signaling cascade, activates the transcription factor NF-kappaB, leading to the expression of proinflammatory cytokines, which trigger inflammation. Such rapid, innate cellular responses serve as the first line of host defense against infection by pathogens, and also stimulate the adaptive immune system to clear the invading microbes. Increasing evidence suggests that TLRs also recognize host-derived ligands, linking this group of PRRs to diseases that may not have an etiology that is associated directly with infections. Advanced glycation end products (AGEs) are nonenzymatically glycated or oxidated proteins, lipids and nucleic acids that are formed in the environment of oxidant stress and hyperglycemia. Binding of AGEs to their receptor RAGE initiates cellular signals that activate NF-kappaB, which results in transcription of proinflammatory factors. RAGE can also interact with other endogenous ligands generated by cell death and tissue injuries. RAGE has been implicated in chronic diseases such as diabetes, atherosclerosis, neurodisorders, cancers, as well as aging. This review discusses the possible role of RAGE as a PRR that may use signaling mechanisms parallel to TLRs', to solicit inflammatory reactions. Thus, in this scenario, RAGE may play a prominent role in the regulation of cellular homeostasis in the context of complex disease progression.

OBJECTIVE

To investigate whether glycoxidation products and the receptor for advanced glycation end products (RAGE) are present and colocalize in subfoveal membranes of patients with age-related macular degeneration (ARMD).

METHODS

Surgically removed subfoveal fibrovascular membranes from 12 patients, 11 related to ARMD and 1 to an idiopathic membrane, were analyzed for the presence of the glycoxidation product N(epsilon)-(carboxymethyl)lysin (CML), one of the receptors for advanced glycation end products, RAGE, and the activation of NFkB, using immunohistochemistry.

RESULTS

CML-like immunoreactivity was found in all ARMD specimens examined adjacent or colocalized with RAGE, but not in the idiopathic membrane. RAGE immunoreactive material was found in CD68-positive cells and in the fibrous matrix. CD68-positive cells and surrounding areas stained for p50, the activated form of NFkB.

CONCLUSIONS

These results indicate that glycoxidation products are present in subretinal membranes of patients with ARMD. The concomitant expression of RAGE in these membranes and the finding of activated NFkB is suggestive of an implication of glycoxidation product formation in the pathogenesis of the disease.

Receptor for advanced glycation endproducts (RAGE), a member of the immunoglobulin superfamily, is a multi-ligand, cell surface receptor expressed by neurons, microglia, astrocytes, cerebral endothelial cells, pericytes, and smooth muscle cells. At least three major types of the RAGE isoforms (full length, C-truncated, and N-truncated) are present in human brains as a result of alternative splicing. Differential expression of each isoform may play a regulatory role in the physiological and pathophysiological functions of RAGE. Analysis of RAGE expression in non-demented and Alzheimer's disease (AD) brains indicated that increases in RAGE protein and percentage of RAGE-expressing microglia paralleled the severity of disease. Ligands for RAGE in AD include amyloid beta peptide (Abeta), S100/calgranulins, advanced glycation endproduct-modified proteins, and amphoterin. Collective evidence from in vitro and in vivo studies supports that RAGE plays multiple roles in the pathogenesis of AD. The major features of RAGE activation in contributing to AD result from its interaction with Abeta, from the positive feedback mechanisms driven by excess amounts of Abeta, and combined with sustained elevated RAGE expression. The adverse consequences of RAGE interaction with Abeta include perturbation of neuronal properties and functions, amplification of glial inflammatory responses, elevation of oxidative stress and amyloidosis, increased Abeta influx at the blood brain barrier and vascular dysfunction, and induction of autoantibodies. In this article, we will review recent advances of RAGE and RAGE activation based on findings from cell cultures, animal models, and human brains. The potential for targeting RAGE mechanisms as therapeutic strategies for AD will be discussed.