Recent clinical studies have reported an increased risk for various types of cancers in patients with diabetes. Diabetes is characterized by increased oxidative stress conditions. Hyperglycemia induces oxidative stress generation in a variety of cells via various metabolic pathways, thus causing oxidative DNA damage, an initial step of carcinogenesis. There is accumulating evidence that advanced glycation end products (AGE), senescent macroprotein derivatives formed at an accelerated rate under normal aging process and diabetes, are involved in the development and progression of cancers. AGE stimulate oxidative stress generation through the interaction with a receptor for AGE (RAGE), while oxidative stress generation promotes the formation of AGE and increases the expression of RAGE. These findings suggest that the crosstalk between the AGE-RAGE system and oxidative stress generation may form a positive feedback loop, thus further increasing the risk for cancers in patients with diabetes. This paper reviews current knowledge about the role of AGE-RAGE system in the development of various types of cancers.

The multi-ligand Receptor for Advanced Glycation Endproducts (RAGE) is implicated in the pathogenesis and progression of chronic diseases such as diabetes and immune/inflammatory disorders. Recent studies are uncovering the precise mechanisms by which distinct RAGE ligands bind the extracellular (soluble) domain of the receptor at the V-, C1- and/or C2-immunoglobulin like domains. Experiments using soluble RAGE in animals as a ligand decoy have illustrated largely beneficial effects in reducing vascular and inflammatory stress and, thereby, preventing long-term tissue damage in models of diabetes and immune/inflammatory disorders. Measurement of soluble RAGE levels in the human, both "total" soluble RAGE and a splice variant-derived product known as endogenous secretory or esRAGE, holds promise for the identification of potential therapeutic targets and/or biomarkers of RAGE activity in disease. In this article, we review the evidence from the rodent to the human implicating RAGE in the diverse disease states in which its ligands accumulate.

The role of hypoxia in regulating tumor progression is still controversial. Here, we demonstrate that, similarly to what previously observed by us in human prostate and breast tumor samples, hypoxia increases expression of the receptor for advanced glycation end products (RAGE) and the purinergic receptor P2X7 (P2X7R). The role of hypoxia was shown by the fact that hypoxia-inducible factor (HIF)-1α silencing downregulated RAGE and P2X7R protein levels as well as nuclear factor-kappaB (NF-κB) expression. In contrast, NF-κB silencing reduced P2X7R expression without affecting RAGE protein levels or nuclear accumulation of HIF-1α. Treatment of hypoxic tumor cells with HMGB1 and BzATP ligands, respectively, of RAGE and P2X7R, activated a signaling pathway that, through Akt and Erk phosphorylation, determines nuclear accumulation of NF-κB and increases cell invasion. Inhibition of Akt by SH5 and Erk by INH1 prevented both nuclear translocation of NF-κB and cell invasion. Moreover, silencing RAGE and P2X7R abolished nuclear accumulation of NF-κB as well as cell invasion without affecting HIF-1α stabilization. Once in the nucleus, NF-κB would contribute to cell survival and invasion under hypoxia, by maintaining RAGE and P2X7R expression levels and matrix metalloproteinases 2 and 9 synthesis. These results show that, hypoxia can upregulate expression levels of membrane receptors that, by binding extracellular molecules eventually released by necrotic cells, contribute to the increased invasiveness of transformed tumor cells. Moreover, these observations strengthen our working hypothesis that upregulation of damage-associated molecular patterns receptors by HIF-1α represents the crucial event bridging hypoxia and inflammation in obtaining the malignant phenotype.

Interaction of advanced glycation end products (AGE) with AGE receptors induces several cellular phenomena potentially relating to diabetic complications. Five AGE receptors identified so far are RAGE (receptor for AGE), galectin-3, 80K-H, OST-48, and SRA (macrophage scavenger receptor class A types I and II). Since SRA is known to belong to the class A scavenger receptor family, and the scavenger receptor collectively represents a family of multiligand lipoprotein receptors, it is possible that CD36, although belonging to the class B scavenger receptor family, can recognize AGE proteins as ligands. This was tested at the cellular level in this study using Chinese hamster ovary (CHO) cells overexpressing human CD36 (CD36-CHO cells). Cellular expression of CD36 was confirmed by immunoblotting and immunofluorescent microscopy using anti-CD36 antibody. Upon incubation at 37 degrees C, (125)I-AGE-bovine serum albumin (AGE-BSA) and (125)I-oxidized low density lipoprotein (LDL), an authentic ligand for CD36, were endocytosed in a dose-dependent fashion and underwent lysosomal degradation by CD36-CHO cells, but not wild-type CHO cells. In binding experiments at 4 degrees C, (125)I-AGE-BSA exhibited specific and saturable binding to CD36-CHO cells (K(d) = 5.6 microg/ml). The endocytic uptake of (125)I-AGE-BSA by these cells was inhibited by 50% by oxidized LDL and by 60% by FA6-152, an anti-CD36 antibody inhibiting cellular binding of oxidized LDL. Our results indicate that CD36 expressed by these cells mediates the endocytic uptake and subsequent intracellular degradation of AGE proteins. Since CD36 is one of the major oxidized LDL receptors and is up-regulated in macrophage- and smooth muscle cell-derived foam cells in human atherosclerotic lesions, these results suggest that, like oxidized LDL, AGE proteins generated in situ are recognized by CD36, which might contribute to the pathogenesis of diabetic macrovascular complications.

OBJECTIVE

To investigate whether advanced glycation end products (AGEs) induce the expression of IL-6 and IL-8 through the receptor for AGEs (RAGE)-activated pathways in human OA chondrocytes.

METHODS

OA chondrocytes were stimulated with AGE-modified BSA (AGE-BSA). Gene expression of IL-6 and IL-8 was quantified by TaqMan assays and the production was determined using ELISAs. Immunoblotting was used to analyse the activation of mitogen-activated protein kinases (MAPKs) and the degradation of IκBα. Activation of NF-κB was determined using an ELISA. Pharmacological studies to elucidate the involved pathways were executed using transfection with small interfering RNAs (siRNAs), inhibitors of MAPKs and NF-κB.

RESULTS

AGE-BSA induced the expression of IL-6 and IL-8 in OA chondrocytes, which was inhibited by pre-treatment with soluble RAGE (sRAGE) or RAGE knockdown by siRNAs. Treatment with SB202190 (p38-MAPK inhibitor) or PD98059 (ERK inhibitor) inhibited AGE-BSA-induced IL-6 and IL-8 expression. However, SP600125 (JNK inhibitor) had no effect on AGE-BSA-induced IL-6 expression but inhibited the expression of IL-8. Treatment with NF-κB inhibitors suppressed AGE-BSA-induced IL-6 and IL-8 expression.

CONCLUSIONS

This is the first study to demonstrate that AGEs induce the expression of IL-6 and IL-8 in OA chondrocytes. A novel finding of our studies is that in OA chondrocytes, AGE-BSA-induced expression of IL-6, but not of IL-8, was independent of the JNK pathway. Activation of NF-κB was an absolute requirement for both IL-6 and IL-8 expression. These results demonstrate that AGE-BSA-induced expression of IL-6 and IL-8 via RAGE is mediated through different MAPK signalling pathways in OA and possibly in other degenerative diseases.

OBJECTIVE

High mobility group box chromosomal protein 1 (HMGB1) is a lately discovered candidate molecule identified as an important extracellular mediator in systemic inflammation. Systemic inflammation results in endothelial cell activation and microvascular injury. In the present study, we investigated the effects of HMGB1 on the activation of human umbilical vein endothelial cells (HUVECs) and defined pathways activated by HMGB1.

METHODS

HUVECs obtained by collagenase treatment of umbilical cord veins were stimulated in vitro with HMGB1. The activation of HUVECs was studied regarding (i) the kinetics of tumor necrosis factor-α (TNF-α) production in HUVECs, (ii) HMGB1-induced up-regulation of receptor for advanced glycation end products (RAGE), (iii) HMGB1-induced nuclear translocation of nuclear factor kappa B (NF-κB) in HUVECs, (iv) the activation of signalling transduction pathways.

RESULTS

HUVECs activation was stimulated by HMGB1 partially in a RAGE-dependent manner. Additionally, the HMGB1-induced activation of HUVECs was significantly inhibited by anti-RAGE monoclonal antibody and Ethyl pyruvate (EP) that had been shown to be an effective anti-inflammatory agent. Short-term prestimulation of HUVECs with HMGB1 caused a time-dependent increase in the secretion of TNF-α and expression of RAGE. Furthermore, HMGB1 stimulation resulted in nuclear translocation of transcription factor NF-κB. Most importantly, pretreatment with anti-RAGE monoclonal antibody significantly decreased the amounts of TNF-α and inhibited the nuclear translocation of NF-κB. Additionally in HUVECs cultures, EP specifically inhibited activation of NF-κB signaling pathway that are critical for TNF-α release.

CONCLUSIONS

In conclusion, Our data present a link between HMGB1and RAGE function of endothelial cells and demonstrate the pathway activated by HMGB1. These findings may provide a novel therapeutic strategy to improve the endothelial cells function.

Metabolic syndrome (MetS) and benign prostatic hyperplasia (BPH)/lower urinary tract symptoms (LUTS) are often associated. One of their common denominators is hypogonadism. However, testosterone supplementation is limited by concerns for potential prostatic side effects. The objective was to determine whether MetS-associated prostate alterations are prevented by testosterone supplementation. We used a previously described animal model of MetS, obtained by feeding male rabbits a high-fat diet (HFD) for 12 weeks. Subsets of HFD rabbits were treated with testosterone or with the farnesoid X receptor agonist INT-747. Rabbits fed a standard diet were used as controls. HFD-animals develop hypogonadism and all the MetS features: hyperglycemia, glucose intolerance, dyslipidemia, hypertension, and visceral obesity. In addition, HFD-animals show a prostate inflammation. Immunohistochemical analysis demonstrated that HFD-induced prostate fibrosis, hypoxia, and inflammation. The mRNA expression of several proinflammatory (IL8, IL6, IL1β, and TNFα), T lymphocyte (CD4, CD8, Tbet, Gata3, and ROR γt), macrophage (TLR2, TLR4, and STAMP2), neutrophil (lactoferrin), inflammation (COX2 and RAGE), and fibrosis/myofibroblast activation (TGFβ, SM22α, αSMA, RhoA, and ROCK1/ROCK2) markers was significantly increased in HFD prostate. Testosterone, as well as INT-747, treatment prevented some MetS features, although only testosterone normalized all the HFD-induced prostate alterations. Interestingly, the ratio between testosterone and estradiol plasma level retains a significant, negative, association with all the fibrosis and the majority of inflammatory markers analyzed. These data highlight that testosterone protects rabbit prostate from MetS-induced prostatic hypoxia, fibrosis, and inflammation, which can play a role toward the development/progression of BPH/LUTS.

BACKGROUND

Huntington's disease (HD) is characterized by a progressive multisystem neuronal atrophy in the brain. Apart from motor signs, cognitive symptoms, particularly executive dysfunctions, are proposed to be recognizable in early stages of disease. The aim of the present study was to clarify if cognitive dysfunction in early stages of HD is correlated with loco-regional structural changes in 3D-MRI.

METHODS

Twenty-five patients with genetically confirmed HD in early clinical stages were included in the study and underwent neuropsychological testing, i.e., the executive tasks Tower of Hanoi (ToH), Stroop Colour Word Interference Test (STROOP), and modified Wisconsin Card Sorting Test (mWCST). High-resolution volume-rendering MRI scans (MP-RAGE) were acquired on a 1.5 T scanner in all patients and were analyzed by statistical parametric mapping and voxel-based morphometry (VBM) in comparison to an age-matched control group.

RESULTS

Group analysis of HD patients demonstrated robust regional decreases of gray matter volumes (p<0.05, corrected for multiple comparisons) in the caudate and the putamen bilaterally with a global maximum at Talairach coordinates 11/4/11 (Z-score=7.06). Executive dysfunction was significantly correlated with the areas of highest significant differences out of VBM results which were located bilaterally in the caudate (ToH: r=0.647, p<0.001; STROOP: r=0.503, p<0.01; mWCST: r=0.452, p<0.05). Moreover, subgroup analyses revealed marked insular atrophy (Talairach coordinates 43/-3/1; Z-score=5.64) in HD patients who performed worse in the single executive tasks.

CONCLUSIONS

Two aspects were most remarkable in this correlational study: (i) striatal atrophy in HD patients in early stages plays an important role not only in impaired motor control but also in executive dysfunction, and (ii) extrastriatal cortical areas, i.e., the insular lobe, seem to be involved in executive dysfunction as assessed by neuropsychological tests requiring for planning and problem solving, stimulus response selectivity and concept formation.

Myocardial infarction, stroke, and venous thromboembolism are characterized by oxygen deprivation. In hypoxia, biological responses are activated that evoke tissue damage. Rapid activation of early growth response-1 in hypoxia upregulates fundamental inflammatory and prothrombotic stress genes. We probed the mechanisms mediating regulation of early growth response-1 and demonstrate that hypoxia stimulates brisk generation of advanced glycation end products (AGEs) by endothelial cells. Via AGE interaction with their chief signaling receptor, RAGE, membrane translocation of protein kinase C-betaII occurs, provoking phosphorylation of c-Jun NH(2)-terminal kinase and increased transcription of early growth response-1 and its downstream target genes. These findings identify RAGE as a master regulator of tissue stress elicited by hypoxia and highlight this receptor as a central therapeutic target to suppress the tissue injury-provoking effects of oxygen deprivation.

The receptor for advanced glycation end-products (RAGE) has been implicated in the pathogenesis of ischemia-reperfusion (I/R) injury in the isolated perfused heart. To test the hypothesis that RAGE-dependent mechanisms modulated responses to I/R in a murine model of transient occlusion and reperfusion of the left anterior descending coronary artery (LAD), we subjected male homozygous RAGE(-/-) mice and their wild-type age-matched littermates to 30 min of occlusion of the LAD followed by reperfusion. At 48 h of reperfusion, hematoxylin and eosin staining revealed significantly larger infarct size in wild-type versus RAGE(-/-) mice. Contractile function, as evaluated by echocardiography 48 h after reperfusion, revealed that fractional shortening was significantly higher in RAGE(-/-) versus wild-type mice. Plasma levels of creatine kinase were markedly decreased in RAGE(-/-) versus wild-type animals. Integral to the impact of RAGE deletion on diminished myocardial damage after infarction was significantly decreased apoptosis in the heart, as assessed by TUNEL staining, release of cytochrome c, and caspase-3 activity. Experiments investigating the impact of RAGE on early signaling pathways influencing myocardial ischemic injury revealed attenuation of JNK and STAT5 phosphorylation in RAGE(-/-) mouse hearts versus robust activation observed in wild-type mice upon ischemia and reperfusion. Solidifying the link to RAGE, these experiments revealed that infarction stimulated the rapid production of advanced glycation end-products in the heart. Thus, we tested the effect of ligand decoy soluble RAGE (sRAGE). Administration of sRAGE protected the myocardium from ischemic damage, similar to the effects observed in RAGE(-/-) mouse hearts. Taken together, these data implicate RAGE and its ligands in the pathogenesis of I/R injury and identify JNK and STAT signal transduction as central downstream effector pathways of the ligand-RAGE axis in the heart subjected to I/R injury.

BACKGROUND

The renin-angiotensin system (RAS) and the accumulation of advanced glycation end products (AGEs) have been implicated in the pathogenesis of diabetic nephropathy. Whether there is a functional interaction between the RAS and AGEs in diabetic nephropathy is not known. In this study, we investigated whether AGEs could activate autocrine angiotensin II (Ang II) signaling and subsequently induce transforming growth factor-beta (TGF-beta)-Smad signaling in cultured rat mesangial cells.

METHODS

The intracellular formation of reactive oxygen species (ROS) was detected using the fluorescent probe CM-H2DCFDA. Ang II was measured by radioimmunoassay. TGF-beta released into media was quantitatively analyzed in an enzyme-linked immunosorbent assay (ELISA). Smad2, p27(Kip1) (p27), fibronectin, and receptor for AGEs (RAGE) protein expression were determined by Western blot analysis. TGF-beta-inducible promoter activity was analyzed by a luciferase assay. DNA synthesis was evaluated by 5-bomo-2'-deoxyuridine (BrdU) incorporation and de novo protein synthesis was determined by [3H]leucine incorporation.

RESULTS

AGEs increased intracellular ROS generation in mesangial cells, and this effect was significantly inhibited by an antiserum against RAGE. AGEs also were found to stimulate Ang II production in a time- and dose-dependent manner, which was completely prevented by an antioxidant, N-acetylcysteine (NAC). AGE-induced TGF-beta overproduction was completely blocked by candesartan, an Ang II type 1 receptor (AT1R) antagonist. Both candesartan and neutralizing antibody against TGF-beta completely prevented AGEs-induced Smad2 phosphorylation and TGF-beta-inducible promoter activity. Furthermore, AGEs were found to inhibit DNA synthesis and to stimulate de novo protein synthesis and fibronectin production in association with up-regulation of p27. All of these phenomena were completely prevented by candesartan or a polyclonal antibody against TGF-beta.

CONCLUSIONS

The present study suggests that AGE-RAGE-mediated ROS generation activates TGF-beta-Smad signaling and subsequently induces mesangial cell hypertrophy and fibronectin synthesis by autocrine production of Ang II. This pathway may provide an important link between metabolic and haemodynamic factors in promoting the development and progression of diabetic nephropathy.

Islet transplantation for the treatment of type 1 diabetes mellitus is limited in its clinical application mainly due to early loss of the transplanted islets, resulting in low transplantation efficiency. NKT cell-dependent IFN-gamma production by Gr-1(+)CD11b(+) cells is essential for this loss, but the upstream events in the process remain undetermined. Here, we have demonstrated that high-mobility group box 1 (HMGB1) plays a crucial role in the initial events of early loss of transplanted islets in a mouse model of diabetes. Pancreatic islets contained abundant HMGB1, which was released into the circulation soon after islet transplantation into the liver. Treatment with an HMGB1-specific antibody prevented the early islet graft loss and inhibited IFN-gamma production by NKT cells and Gr-1(+)CD11b(+) cells. Moreover, mice lacking either of the known HMGB1 receptors TLR2 or receptor for advanced glycation end products (RAGE), but not the known HMGB1 receptor TLR4, failed to exhibit early islet graft loss. Mechanistically, HMGB1 stimulated hepatic mononuclear cells (MNCs) in vivo and in vitro; in particular, it upregulated CD40 expression and enhanced IL-12 production by DCs, leading to NKT cell activation and subsequent NKT cell-dependent augmented IFN-gamma production by Gr-1(+)CD11b(+) cells. Thus, treatment with either IL-12- or CD40L-specific antibody prevented the early islet graft loss. These findings indicate that the HMGB1-mediated pathway eliciting early islet loss is a potential target for intervention to improve the efficiency of islet transplantation.

The multiligand receptor for advanced glycation end products (RAGE) of the Ig superfamily transduces the biological impact of discrete families of ligands, including advanced glycation end products, certain members of the S100/calgranulin family, high mobility group box-1, Mac-1 (alpha(M)beta(2), CD11b/CD18), and amyloid-beta peptide and beta-sheet fibrils. Although structurally dissimilar, at least at the monomeric level, recent evidence suggests that oligomeric forms of these RAGE ligands may be especially apt to activate the receptor and up-regulate a program of inflammatory and tissue injury-provoking genes. The challenge in probing the biology of RAGE and its impact in acute responses to stress and the potential development of chronic disease is to draw the line between mechanisms that evoke repair versus those that sustain inflammation and tissue damage. In this review, we suggest the concept that the ligands of RAGE comprise a primal program in the acute response to stress. When up-regulated in environments laden with oxidative stress, inflammation, innate aging, or high glucose, as examples, the function of these ligand families may be transformed from ones linked to rapid repair to those that drive chronic disease. Identification of the threshold beyond which ligands of RAGE mediate repair versus injury is a central component in delineating optimal strategies to target RAGE in the clinic.

Accumulation of β-amyloid and hyperphosphorylated tau with synapse damage and memory deterioration are hallmark lesions of Alzheimer disease (AD), but the upstream causative factors are elusive. The advanced glycation endproducts (AGEs) are elevated in AD brains and the AGEs can stimulate β-amyloid production. Whether and how AGEs may cause AD-like tau hyperphosphorylation and memory-related deficits is not known. Here we report that AGEs induce tau hyperphosphorylation, memory deterioration, decline of synaptic proteins, and impairment of long-term potentiation (LTP) in rats. In SK-NS-H cells, upregulation of AGEs receptor (RAGE), inhibition of Akt, and activation of glycogen synthase kinase-3 (GSK-3), Erk1/2, and p38 were observed after treatment with AGEs. In rats, blockage of RAGE attenuated the AGE-induced GSK-3 activation, tau hyperphosphorylation, and memory deficit with restoration of synaptic functions, and simultaneous inhibition of GSK-3 also antagonized the AGE-induced impairments. Our data reveal that AGEs can induce tau hyperphosphorylation and impair synapse and memory through RAGE-mediated GSK-3 activation and targeting RAGE/GSK-3 pathway can efficiently improve the AD-like histopathological changes and memory deterioration.

Amphoterin (HMG1) is a 30-kD heparin-binding protein which is functionally associated with the outgrowth of cytoplasmic processes in developing neurones. Amphoterin has been shown to mediate adhesive and proteolytic interactions at the leading edge of motile cells. Recently it was shown that inhibition of amphoterin interactions with its cell surface receptor (RAGE) suppresses tumour growth and metastasis. In this work we have identified amphoterin polypeptide and its mRNA in human platelets. Amphoterin had a cytoplasmic localisation in resting platelets according to subcellular fractionation studies and immunogold electronmicroscopy. After platelet activation, part of amphoterin was associated with the external surface of plasma membrane. Externalisation of amphoterin during platelet activation was also detected in immunofluorescence studies. Amphoterin was detectable in human serum (0.2 ng/ml) but not in plasma. Resting platelets treated with PGI2 and forskolin bound to immobilised recombinant amphoterin independently of divalent cations. The binding induced a spicular morphology in platelets, and was effectively inhibited by heparin. Amphoterin-binding protein components on the platelet surface were not identified, but amphoterin bound to phosphatidylserine and sulfatide in lipid binding assays. Our results suggest that amphoterin is an endogenous protein in human platelets, which is exported to the cell surface during platelet activation. Interaction of amphoterin with the platelet surface may be mediated by sulfoglycolipids and phospholipids.

The receptor for advanced glycation end products (RAGE) is known to be causally involved in a variety of pathophysiological processes, e.g. immune/inflammatory disorders, Alzheimer disease, tumors, and abnormalities associated with diabetes as arteriosclerosis or disordered wound healing. So far, human cDNAs have been characterized encoding for the RAGE receptor and a truncated soluble form lacking the transmembrane and the cytosolic domain. The latter form represents a naturally occurring competitive inhibitor of signalling pathways induced by the membrane-standing RAGE receptor. In order to perform a relative expression analysis of both RAGE forms, an RT-PCR experiment was designed allowing the simultaneous amplification of corresponding transcripts. We were able to identify three novel human RAGE transcripts all encoding truncated soluble forms of RAGE. The relative expression ratios for the full-length RAGE transcript to the sum of its splice-variants encoding the soluble variants varied strongly among the tissues tested. Therefore, the pre-mRNA of RAGE must be subject to regulated alternative splicing activated by extracellular cues of yet unknown cellular signalling pathways. Thus, as deduced from the occurrence at the RNA level, it can be hypothesized that there is a complex RAGE regulation network involving isoforms competing for the binding of ligands.

Glycation is the nonenzymatic reaction of glucose, alpha-oxoaldehydes, and other saccharide derivatives with proteins, nucleotides, and lipids. Early glycation adducts (fructosamines) and advanced glycation adducts (AGEs) are formed. "Glycoxidation" is a term used for glycation processes involving oxidation. Sural, peroneal, and saphenous nerves of human diabetic subjects contained AGEs in the perineurium, endothelial cells, and pericytes of endoneurial microvessels and in myelinated and unmyelinated fibres localized to irregular aggregates in the cytoplasm and interstitial collagen and basement membranes. Pentosidine content was increased in cytoskeletal and myelin protein extracts of the sural nerve of human subjects and cytoskeletal proteins of the sciatic nerve of streptozotocin-induced diabetic rats. AGEs in the sciatic nerve of diabetic rats were decreased by islet transplantation. Improved glycemic control of diabetic patients may be expected to decrease protein glycation in the nerve. Protein glycation may decrease cytoskeletal assembly, induce protein aggregation, and provide ligands for cells surface receptors. The receptor for advanced glycation and products (RAGE) was expressed in peripheral neurons. It is probable that high intracellular glucose concentration is an important trigger for increased glycation, leading to increased formation of methylglyoxal, glyoxal, and 3-deoxyglucosone that glycate proteins to form AGEs intracellularly and extracellularly. Oxidative stress enhances these processes and is, in turn, enhanced by AGE/RAGE interactions. An established therapeutic strategy to prevent glycation is the use of alpha-oxoaldehyde scavengers. Available therapeutic options for trial are high-dose nicotinamide and thiamine therapies to prevent methylglyoxal formation. Future possible therapeutic strategies are RAGE antagonists and inducers of the enzymatic antiglycation defense. More research is required to understand the role of glycation in the development of diabetic neuropathy.

The transmembrane receptor (RAGE) of advanced glycation endproducts (AGEs), is abundantly present in the lung. Although the interaction of AGEs and RAGE plays an important role in vasculopathies, particularly in diabetes, the lung is not a classical target organ of diabetes. Thus, the role of RAGE in the lung is still obscure. This study sought to precisely localise RAGE in the lungs of rat and human by immunohistochemistry, double immunofluorescence and immunoelectron microscopy using a polyclonal antiserum developed against human recombinant RAGE. Anti-RAGE immunoreactivity was prominent in alveolar epithelial type I pneumocytes, while it was absent from type II pneumocytes and capillary endothelium. Cell type specificity was demonstrated by colocalisation with well established cell markers. Quantitative immunoelectron microscopy of cryo-substituted, Lowicryl-embedded rat and human specimens demonstrated a unique labelling pattern of RAGE in that it selectively localised to the basal cell membrane of type I pneumocytes. Labelling pattern was independent of the mode of fixation. Equivalent labelling densities were calculated from a fibrotic rat lung 3 months after irradiation. This highly selective localisation of RAGE to the basal face of type I pneumocytes and its absence from capillary endothelium might explain the resistance of the lung to typical diabetic complications.

An important component of amyloid fibrils in dialysis-related amyloidosis is a form of beta2microglobulin modified with advanced glycation end products (AGEs) of the Maillard reaction, known as AGE-beta2M. We demonstrate here that the interaction of AGE-beta2M with mononuclear phagocytes (MPs), cells important in the pathogenesis of the inflammatory arthropathy of dialysis-related amyloidosis, is mediated by the receptor for AGEs, or RAGE. 125I-AGE-beta2M bound to immobilized RAGE or to MPs in a specific, dose-dependent manner (Kd approximately 53.5 and approximately 81.6 nM, respectively), a process inhibited in the presence of RAGE blockade. AGE-beta2M-mediated monocyte chemotaxis was prevented by excess sRAGE or anti-RAGE IgG. Induction of tumor necrosis factor-alpha (TNF) expression by MPs exposed to AGE-beta2M resulted from engagement of RAGE, as appearances of TNF transcripts and TNF antigen release into culture supernatants were prevented by addition of sRAGE, a process mediated, at least in part, by oxidant stress. AGE-beta2M reduced cytochrome c and the elaboration of TNF by MPs was inhibited by N-acetylcysteine. Consistent with these data, immunohistochemical studies of AGE-laden amyloid deposits of a long-term hemodialysis patient revealed positive staining for RAGE in the MPs infiltrating these lesions. These data indicate that RAGE is a central binding site for AGEs formed in vivo and suggest that AGE-beta2M-MP-RAGE interaction likely contributes to the initiation of an inflammatory response in amyloid deposits of long-term hemodialysis patients, a process which may ultimately lead to bone and joint destruction.

BACKGROUND

Advanced glycation end products (AGEs) accumulate in patients with decreased renal function and exert various toxic effects through the receptor for AGEs (RAGE). Soluble RAGE (sRAGE) is a naturally occurring inhibitor of AGE-RAGE action. The aim of the study is to describe the relationship of sRAGE to renal function and dialysis modalities.

METHODS

The studied group consisted of 81 patients: 25 patients with various degrees of decreased renal function, 20 long-term hemodialysis (HD) patients, 15 peritoneal dialysis (PD) patients, and 21 healthy age-matched subjects. sRAGE was assessed immunochemically (enzyme-linked immunosorbent assay), and routine biochemical parameters were measured by means of certified methods.

RESULTS

sRAGE level correlates positively with serum creatinine concentration (r = 0.50; P < 0.05), and its relationship to creatinine clearance is hyperbolic. sRAGE levels are elevated significantly, mainly in patients with end-stage renal disease (3,119.0 +/- 968.4 pg/mL in HD patients and 3,652.7 +/- 1,677.7 pg/mL in PD patients versus 1,405.1 +/- 426.1 pg/mL in controls; both P < 0.001 versus controls). In PD patients, sRAGE is detectable in spent dialysate (median, 75.8 pg/mL), correlates with its serum levels (r = 0.67; P < 0.05), and is related to protein losses in dialysate. In HD patients, sRAGE levels increase by 50% (P < 0.001) from 0 to 15 minutes during both HD and hemodiafiltration, and then decrease until the end of the session.

CONCLUSIONS

Serum sRAGE levels increase in patients with decreased renal function, mainly patients with end-stage renal disease. It remains to be elucidated whether the increase is caused just by decreased renal function or whether sRAGE is upregulated to protect against toxic effects of AGEs.

The ligand - receptor for advanced glycation end-products (RAGE) axis has emerged as a novel pathway involved in a wide spectrum of diseases, including diabetes mellitus, atherothrombosis, chronic renal failure, rheumatoid arthritis, neurodegeneration, cancer and aging. Circulating soluble forms of RAGE (sRAGE), arising from receptor ectodomain shedding and splice variant [endogenous secretory (es) RAGE] secretion, may counteract RAGE-mediated pathogenesis, by acting as a decoy. Several studies suggest that decreased levels of sRAGE and/or esRAGE may be useful as a biomarker of ligand-RAGE pathway hyperactivity and inadequate endogenous protective response, thus providing a powerful complement to cardiovascular risk stratification and an interesting target of therapeutic interventions. This review will focus on the pathophysiological determinants of soluble forms of RAGE in different clinical settings, with particular reference to the mechanisms involved in their generation and clearance, the association with cardiovascular risk factors, the interplay with low-grade inflammation, oxidative stress and endothelial dysfunction, and the possible pharmacological modulation of their plasma levels.

The alternative splicing of pre-mRNAs is a critical mechanism in genomic complexity, disease, and development. Studies of the receptor for advanced glycation end-products (RAGE) indicate that this gene undergoes a variety of splice events in humans. However, no studies have extensively analyzed the tissue distribution in other species or compared evolutionary differences of RAGE isoforms. Because the majority of studies probing RAGE function have been performed in murine models, we therefore performed studies to identify and characterize the splice variants of the murine RAGE gene, and we compared these to human isoforms. Here, using mouse tissues, we identified numerous splice variants including changes in the extracellular domain or the removal of the transmembrane and cytoplasmic domains, which produce soluble splice isoforms. Comparison of splice variants between humans and mice revealed homologous regions in the RAGE gene that undergo splicing as well as key species-specific mechanisms of splicing. Further analysis of tissue splice variant distribution in mice revealed major differences between lung, kidney, heart, and brain. To probe the potential impact of disease-like pathological states, we studied diabetic mice and report that RAGE splice variation changed dramatically, resulting in an increase in production of soluble RAGE (sRAGE) splice variants, which were not associated with detectable levels of sRAGE in murine plasma. In conclusion, we have determined that the murine RAGE gene undergoes extensive splicing with distinct splice isoforms being uniquely distributed in different tissues. These differences in RAGE splicing in both physiological and pathogenic states further expand our understanding of the biological repertoire of this receptor in health and disease.

Endothelial progenitor cells (EPCs) play an important role in preventing atherosclerosis. The factors that regulate the function of EPCs are not completely clear. Increased formation of advanced glycation endproducts (AGEs) is generally regarded as one of the main mechanisms responsible for vascular damage in patients with diabetes and atherosclerosis. AGEs lead to the generation of reactive oxygen species (ROS) and part of the regenerative capacity of EPCs seems to be due to their low baseline ROS levels and reduced sensitivity to ROS-induced cell apoptosis. Therefore, we tested the hypothesis that AGEs can alter functions and promote apoptosis in EPCs through overpress cell oxidant stress. EPCs, isolated from bone marrow, were cultured in the absence or presence of AGEs (50, 100, and 200 microg/ml). A modified Boyden's chamber was used to assess the migration of EPCs and the number of recultured EPCs was counted to measure the adhesiveness function. MTT assay was used to determine the proliferation function. ROS were analyzed using the ROS assay kit. A spectrophotometer was used to assess superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) activity, and PCR was used to test mRNA expression of SOD and GSH-PX. SiRNA was used to block receptor for advanced glycation endproducts (RAGEs) expression. Apoptosis was evaluated by Annexin V immunostaining and TUNEL staining. Co-culturing with AGEs increases ROS production, decreases anti-oxidant defenses, overpresses oxidant stress, inhibits the proliferation, migration, and adhesion of EPCs, and induces EPCs apoptosis. In addition, these effects were attenuated during block RAGE protein expression by siRNA. AGEs may serve to impair EPCs functions through RAGE-mediate oxidant stress, and promote EPCs sensitivity toward oxidative-stress-mediated apoptosis, which indicates a new pathophysiological mechanism of disturbed vascular adaptation in atherosclerosis and suggests that lower levels of AGEs might improve the success of progenitor cell therapy.

BACKGROUND

Receptor for advanced-glycation end products (RAGE) and its ligands AGEs and S100/calgranulins have been implicated in a range of disorders. However, the role of RAGE/ligand interaction in neointimal hyperplasia after vascular injury remains unclear.

RESULTS

We examined the expression of RAGE and its ligands after balloon injury of the carotid artery in both Zucker diabetic and nondiabetic rats. Using a soluble portion of the extracellular domain of RAGE, we determined the effects of suppressing RAGE/ligand interaction on vascular smooth muscle cell (VSMC) proliferation and neointimal formation after arterial injury. We demonstrate a significantly increased accumulation of AGE and immunoreactivities of RAGE and S100/calgranulins in response to balloon injury in diabetic compared with nondiabetic rats. Blockade of RAGE/ligand interaction significantly decreased S100-stimulated VSMC proliferation in vitro and bromodeoxyuridine (BrdU)-labeled proliferating VSMC in vivo, and suppressed neointimal formation and increased luminal area in both Zucker diabetic and nondiabetic rats.

CONCLUSIONS

These findings indicate that RAGE/ligand interaction plays a key role in neointimal formation after vascular injury irrespective of diabetes status and suggest a novel target to minimize neointimal hyperplasia.