Activation Of Receptor For Advanced Glycation Endproducts Research Articles

Transactivation of RAGE mediates angiotensin-induced inflammation and atherogenesis.

Activation of the type 1 angiotensin II receptor (AT1) triggers proinflammatory signaling through pathways independent of classical Gq signaling that regulate vascular homeostasis. Here, we report that the AT1 receptor preformed a heteromeric complex with the receptor for advanced glycation endproducts (RAGE). Activation of the AT1 receptor by angiotensin II (Ang II) triggered transactivation of the cytosolic tail of RAGE and NF-κB-driven proinflammatory gene expression independently of the liberation of RAGE ligands or the ligand-binding ectodomain of RAGE. The importance of this transactivation pathway was demonstrated by our finding that adverse proinflammatory signaling events induced by AT1 receptor activation were attenuated when RAGE was deleted or transactivation of its cytosolic tail was inhibited. At the same time, classical homeostatic Gq signaling pathways were unaffected by RAGE deletion or inhibition. These data position RAGE transactivation by the AT1 receptor as a target for vasculoprotective interventions. As proof of concept, we showed that treatment with the mutant RAGE peptide S391A-RAGE362-404 was able to inhibit transactivation of RAGE and attenuate Ang II-dependent inflammation and atherogenesis. Furthermore, treatment with WT RAGE362-404 restored Ang II-dependent atherogenesis in Ager/Apoe-KO mice, without restoring ligand-mediated signaling via RAGE, suggesting that the major effector of RAGE activation was its transactivation.

SRAGE attenuates angiotensin II-induced cardiomyocyte hypertrophy by inhibiting RAGE-NFκB-NLRP3 activation.

The receptor for advanced glycation endproducts (RAGE) is an innate immunity receptor that has been implicated in the pathogenesis of atherosclerotic cardiovascular disease. However, the possibility that RAGE-mediated signaling is involved in angiotensin II (Ang II)-induced cardiac left ventricular hypertrophy has yet to be investigated. We therefore determined whether RAGE has a role in regulating pathological cardiac hypertrophy. Protein abundance was estimated using Western blotting and intracellular ROS level and phospho-p65 were detected using fluorescence microscopy. Enzyme-linked immunosorbent assay was used to detect HMGB1 and IL-1β. All in vitro experiments were performed using H9C2 cells. To induce cardiomyocyte hypertrophy, 300nM Ang II was treated for 48h and 2µg/ml sRAGE was treated 1h prior to addition of Ang II. sRAGE attenuated Ang II-induced cardiomyocyte hypertrophy by downregulating RAGE and angiotensin II type 1 receptor expression. Secretion levels of high motility group box 1 and interleukin-1β, estimated from a cell culture medium, were significantly reduced by sRAGE. Activated PKCs and ERK1/2, important signals in left ventricular hypertrophy (LVH) development, were downregulated by sRAGE treatment. Furthermore, we found that nuclear factor-κB and NOD-like receptor protein 3 (NLRP3) were associated with RAGE-mediated cardiomyocyte hypertrophy. In the context of these results, we conclude that RAGE induces cardiac hypertrophy through the activation of the PKCs-ERK1/2 and NF-κB-NLRP3-IL1β signaling pathway, and suggest that RAGE-NLRP3 may be an important mediator of Ang II-induced cardiomyocyte hypertrophy. In addition, we determined that inhibition of RAGE activation with soluble RAGE (sRAGE) has a protective effect on Ang II-induced cardiomyocyte hypertrophy.

The advanced glycation end-product Nϵ -carboxymethyllysine promotes progression of pancreatic cancer: implications for diabetes-associated risk and its prevention.

Diabetes is an established risk factor for pancreatic cancer (PaC), together with obesity, a Western diet, and tobacco smoking. The common mechanistic link might be the accumulation of advanced glycation end-products (AGEs), which characterizes all of the above disease conditions and unhealthy habits. Surprisingly, however, the role of AGEs in PaC has not been examined yet, despite the evidence of a tumour-promoting role of receptor for advanced glycation end-products (RAGE), the receptor for AGEs. Here, we tested the hypothesis that AGEs promote PaC through RAGE activation. To this end, we investigated the effects of the AGE Nϵ -carboxymethyllysine (CML) in human pancreatic ductal adenocarcinoma (PDA) cell lines and in a mouse model of Kras-driven PaC interbred with a bioluminescent model of proliferation. Tumour growth was monitored in vivo by bioluminescence imaging and confirmed by histology. CML promoted PDA cell growth and RAGE expression, in a concentration-dependent and time-dependent manner, and activated downstream tumourigenic signalling pathways. These effects were counteracted by RAGE antagonist peptide (RAP). Exogenous AGE administration to PaC-prone mice induced RAGE upregulation in pancreatic intraepithelial neoplasias (PanINs) and markedly accelerated progression to invasive PaC. At 11 weeks of age (6 weeks of CML treatment), PaC was observed in eight of 11 (72.7%) CML-treated versus one of 11 (9.1%) vehicle-treated [control (Ctr)] mice. RAP delayed PanIN development in Ctr mice but failed to prevent PaC promotion in CML-treated mice, probably because of competition with soluble RAGE for binding to AGEs and/or compensatory upregulation of the RAGE homologue CD166/ activated leukocyte cell adhesion molecule, which also favoured tumour spread. These findings indicate that AGEs modulate the development and progression of PaC through receptor-mediated mechanisms, and might be responsible for the additional risk conferred by diabetes and other conditions characterized by increased AGE accumulation. Finally, our data suggest that an AGE reduction strategy, instead of RAGE inhibition, might be suitable for the risk management and prevention of PaC. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

RAGE-induced changes in the proteome of alveolar epithelial cells

The receptor for advanced glycation end-products (RAGE) is a pattern recognition receptor and member of the immunoglobulin superfamily. RAGE is constitutively expressed in the distal lung where it co-localizes with the alveolar epithelium; RAGE expression is otherwise minimal or absent, except with disease. This suggests RAGE plays a role in lung physiology and pathology. We used proteomics to identify and characterize the effects of RAGE on rat alveolar epithelial (R3/1) cells. LC-MS/MS identified 177 differentially expressed proteins and the PANTHER Classification System further segregated proteins. Proteins involved in gene transcription (RNA and mRNA splicing, mRNA processing) and transport (protein, intracellular protein) were overrepresented; genes involved in a response to stimulus were underrepresented. Immune system processes and response to stimuli were downregulated with RAGE knockdown. Western blot confirmed RAGE-dependent changes in protein expression for NFκB and NLRP3 that was functionally supported by a reduction in IL-1β and phosphorylated p65. We also assessed RAGE's effect on redox regulation and report that RAGE knockdown attenuated oxidant production, decreased protein oxidation, and increased reduced thiol pools. Collectively the data suggest that RAGE is a critical regulator of epithelial cell response and has implications for our understanding of lung disease, specifically acute lung injury. Significance statementIn the present study, we undertook the first proteomic evaluation of RAGE-dependent processes in alveolar epithelial cells. The alveolar epithelium is a primary target during acute lung injury, and our data support a role for RAGE in gene transcription, protein transport, and response to stimuli. More over our data suggest that RAGE is a critical driver of redox regulation in the alveolar epithelium. The conclusions of the present work assist to unravel the molecular events that underlie the function of RAGE in alveolar epithelial cells and have implications for our understanding of RAGE signaling during lung injury. Our study was the first proteomic comparison showing the effects of RAGE activation from alveolar epithelial cells that constitutively express RAGE and these results can affect a wide field of lung biology, pulmonary therapeutics, and proteomics.

Soluble Receptor for Advanced Glycation End Products Improves Stromal Cell–Derived Factor-1 Activity in Model Diabetic Environments

Objective: In diabetes, hyperglycemia causes the accumulation of advanced glycation end products (AGEs) that trigger reactive oxygen species (ROS) generation through binding the receptor for AGEs (RAGE). Because exogenous growth factors have had little success in enhancing chronic wound healing, we investigated whether hyperglycemia-induced AGEs interfere with cellular responses to extracellular signals. We used stromal cell-derived factor-1 (SDF-1), an angiogenic chemokine also known to promote stem cell recruitment in skin wounds. Approach: Human leukemia-60 (HL-60) cells and mouse peripheral blood mononuclear cells (PBMCs), which express the SDF-1 receptor CXCR-4, were incubated for 24 h in medium supplemented with 25 mM d-glucose. Soluble RAGE (sRAGE) was used to block RAGE activation. Response to SDF-1 was measured in cellular migration and ROS assays. A diabetic murine excisional wound model measured SDF-1 liposome and sRAGE activity in vivo. Results: Hyperglycemia led to significant accumulation of AGEs, decreased SDF-1-directed migration, and elevated baseline ROS levels; it suppressed the ROS spike normally triggered by SDF-1. sRAGE decreased the ROS baseline and restored both the SDF-1-mediated spike and cell migration. Topically applied sRAGE alone promoted healing and enhanced the effect of exogenous SDF-1 on diabetic murine wounds. Innovation: While there is interest in using growth factors to improve wound healing, this strategy is largely ineffective in diabetic wounds. We show that sRAGE may restore signaling, thus potentiating the effect of exogenously applied growth factors. Conclusion: Blocking RAGE with sRAGE restores SDF-1-mediated cellular responses in hyperglycemic environments and may potentiate the effectiveness of SDF-1 applied in vivo.

Advanced glycation end products mediated cellular and molecular events in the pathology of diabetic nephropathy.

Diabetic nephropathy (DN) is a major cause of morbidity and mortality in diabetic patients and a leading cause of end-stage renal disease (ESRD). Degenerative changes such as glomerular hypertrophy, hyperfiltration, widening of basement membranes, tubulointerstitial fibrosis, glomerulosclerosis and podocytopathy manifest in various degrees of proteinuria in DN. One of the key mechanisms implicated in the pathogenesis of DN is non-enzymatic glycation (NEG). NEG is the irreversible attachment of reducing sugars onto free amino groups of proteins by a series of events, which include the formation of Schiff's base and an Amadori product to yield advanced glycation end products (AGEs). AGE modification of client proteins from the extracellular matrix induces crosslinking, which is often associated with thickening of the basement membrane. AGEs activate several intracellular signaling cascades upon interaction with receptor for AGEs (RAGE), which manifest in aberrant cellular responses such as inflammation, apoptosis and autophagy, whereas other receptors such as AGE-R1, AGE-R3 and scavenger receptors also bind to AGEs and ensue endocytosis and degradation of AGEs. Elevated levels of both serum and tissue AGEs are associated with adverse renal outcome. Increased evidence supports that attenuation of AGE formation and/or inhibition of RAGE activation manifest(s) in improving renal function. This review provides insights of NEG, discusses the cellular and molecular events triggered by AGEs, which manifest in the pathogenesis of DN including renal fibrosis, podocyte epithelial-mesenchymal transition and activation of renin-angiotensin system. Therapies designed to target AGEs, such as inhibitors of AGEs formation and crosslink breakers, are discussed.

Inhibition of the Receptor for Advanced Glycation End-Products (RAGE) Attenuates Neuroinflammation While Sensitizing Cortical Neurons Towards Death in Experimental Subarachnoid Hemorrhage.

Subarachnoid hemorrhage (SAH) is a threatening and devastating neurological insult with high mortality and morbidity rates. Despite considerable efforts, the underlying pathophysiological mechanisms are still poorly understood. The receptor for advanced glycation end products (RAGE) is a multiligand receptor that has been implicated in various pathological conditions. We previously showed that RAGE was upregulated and may be involved in pathophysiology of SAH. In the current study, we investigated its potential role in SAH. We found that the upregulation of RAGE after SAH was NF-κB-dependent positive feedback regulation. Further, pharmacological inhibition of RAGE attenuated neuroinflammation, indicating a possible contributive role of RAGE in inflammation-associated brain injury after SAH. Conversely, however, inhibition of RAGE sensitized neurons, exacerbating cell death, which correlated with augmented apoptosis and diminished autophagy, suggesting that activation of RAGE may protect against SAH-induced neuronal injury. Furthermore, we demonstrate that inhibition of RAGE significantly reduced brain edema and improved neurological function at day 1 but not at day 3 post-SAH. Taken together, these results suggest that RAGE exerts dual role after SAH. Our findings also suggest caution should be exercised in setting RAGE-targeted treatment for SAH.

IRAGE as a novel carboxymethylated peptide that prevents advanced glycation end product-induced apoptosis and endoplasmic reticulum stress in vascular smooth muscle cells

Advanced glycation end-products (AGE) and the receptor for AGE (RAGE) have been linked to numerous diabetic vascular complications. RAGE activation promotes a self-sustaining state of chronic inflammation and has been shown to induce apoptosis in various cell types. Although previous studies in vascular smooth muscle cells (VSMC) showed that RAGE activation increases vascular calcification and interferes with their contractile phenotype, little is known on the potential of RAGE to induce apoptosis in VSMC. Using a combination of apoptotic assays, we showed that RAGE stimulation with its ligand CML-HSA promotes apoptosis of VSMC. The formation of stress granules and the increase in the level of the associated protein HuR point toward RAGE-dependent endoplasmic reticulum (ER) stress, which is proposed as a key contributor of RAGE-induced apoptosis in VSMC as it has been shown to promote cell death via numerous mechanisms, including up-regulation of caspase-9. Chronic NF-κB activation and modulation of Bcl-2 homologs are also suspected to contribute to RAGE-dependent apoptosis in VSMC. With the goal of reducing RAGE signaling and its detrimental impact on VSMC, we designed a RAGE antagonist (iRAGE) derived from the primary amino acid sequence of HSA. The resulting CML peptide was selected for the high glycation frequency of the primary sequence in the native protein in vivo. Pretreatment with iRAGE blocked 69.6% of the increase in NF-κB signaling caused by RAGE activation with CML-HSA after 48h. Preincubation with iRAGE was successful in reducing RAGE-induced apoptosis, as seen through enhanced cell survival by SPR and reduced PARP cleavage. Activation of executioner caspases was 63.5% lower in cells treated with iRAGE before stimulation with CML-HSA. To our knowledge, iRAGE is the first antagonist shown to block AGE-RAGE interaction and we propose the molecule as an initial candidate for drug discovery.

The Receptor for Advanced Glycation Endproducts Does Not Contribute to Pathology in a Mouse Mesenteric Ischemia/Reperfusion-Induced Injury Model.

The receptor for advanced glycation endproducts (RAGE) can engage a diverse class of ligands and contribute to the immune and inflammatory response to infection and injury. It is known to be a pathogenic receptor in many inflammatory diseases, including ischemia/reperfusion (IR) injuries in several tissues; however, its role has not been investigated in IR injuries of the intestine to date. Mesenteric (or intestinal) IR leads to recruitment of inflammatory cells into intestinal interstitial spaces, which markedly disrupts intestinal mucosa. IR-induced mucosal injury is accompanied by the development of a local and systemic inflammatory response and remote organ injury, and results in high mortality in the clinic. We hypothesized that elimination of RAGE signaling using RAGE−/− mice would result in decreased local and remote organ injury and reduced inflammation in a mesenteric IR model, and thus be a target for therapeutic intervention. We found that RAGE ligands including HMGB-1 and C3a were elevated after mesenteric IR indicating the potential for enhanced RAGE activation in this model. However despite this, wild-type and RAGE−/− mice both displayed similar degrees of mesenteric injury, neutrophil infiltration, intestinal edema, cytokine generation, neutrophil mobilization, and remote organ injury after mesenteric IR. We, therefore, conclude that despite its role in other organ IR injuries, and the robust production of RAGE ligands after intestinal ischemia, RAGE itself does not directly influence tissue injury and the inflammatory response in mesenteric IR.

HMGB1 enhances the protumoral activities of M2 macrophages by a RAGE-dependent mechanism.

The monocyte-macrophage lineage shows a high degree of diversity and plasticity. Once they infiltrate tissues, they may acquire two main functional phenotypes, being known as the classically activated type 1 macrophages (M1) and the alternative activated type 2 macrophages (M2). The M1 phenotype can be induced by bacterial products and interferon-γ and exerts a cytotoxic effect on cancer cells. Conversely, the alternatively activated M2 phenotype is induced by Il-4/IL13 and promotes tumor cell growth and vascularization. Although receptor for advanced glycation end-products (RAGE) engagement in M1 macrophages has been reported by several groups to promote inflammation, nothing is known about the functionality of RAGE in M2 macrophages. In the current study, we demonstrate that RAGE is equally expressed in both macrophage phenotypes and that RAGE activation by high-mobility group protein box1 (HMGB1) promotes protumoral activities of M2 macrophages. MKN45 cells co-cultured with M2 macrophages treated with HMGB1 at different times displayed higher invasive abilities. Additionally, conditioned medium from HMGB1-treated M2 macrophages promotes angiogenesis in vitro. RAGE-targeting knockdown abrogates these activities. Overall, the present findings suggest that HMGB1 may contribute, by a RAGE-dependent mechanism, to the protumoral activities of the M2 phenotype.

Differential Placental Expression of the Receptor for Advanced Glycation End‐Products (RAGE) in Normal and Complicated Pregnancies

Receptor for Advanced Glycation End‐Products (RAGE) is a cell surface receptor that modulates inflammatory pathways. Inflammation is associated with the development of pregnancy pathologies such as preeclampsia (PE), intrauterine growth restriction (IUGR), gestational diabetes mellitus (GDM), and pre‐term labor (PTL) that can lead to maternal and fetal complications. Our objective was to examine placental RAGE expression to implicate RAGE signaling in these complications. We performed qPCR, western blotting and immunofluorescence to determine levels of RAGE gene activation, protein expression and RAGE localization in the placenta. An ELISA was used to determine TNF‐α secretion. First trimester trophoblast cells were used to examine RAGE expression and downstream signaling molecules after RAGE activation by the ligand AGEs. Compared to control placental samples we observed: 1) increased localization of RAGE to the PE placental trophoblast; 2) decreased RAGE localization in the IUGR placenta; 3) a 1.5 fold increase (p<0.05) in placental RAGE gene expression during IUGR; 3) a 2‐fold increase (p<0.03) of placental RAGE protein expression in PE/IUGR; and 3) a 2‐fold decrease (p<0.05) in placental RAGE protein expression during IUGR. In first trimester cell lines we observed: 1) an 8‐fold increase in p‐ERK expression after 25ug treatment of AGES (p<0.007) and 2) a 1.8‐fold increase (p<0.05) of TNF‐α secretion after 25ug treatment of AGEs, a common RAGE ligand. We conclude that placental RAGE is activated in PE/IUGR situations and that RAGE‐mediated inflammation in the trophoblast includes increased TNF‐α secretion through the activation of ERK signaling.

Role of receptor for advanced glycation end products (RAGE) and its ligands in cancer risk.

Reducing sugars can react non-enzymatically with the amino groups of proteins, lipids, and nucleic acids to initiate a complex series of rearrangements and dehydrations, and then to produce a class of irreversibly cross-linked heterogeneous fluorescent moieties, termed advanced glycation end products (AGEs). The formation and accumulation of AGEs in numerous tissues have been known to progress in a normal aging process and at an accelerated rate under hyperglycemic and/or oxidative stress conditions. There is a growing body of evidence that interaction of AGEs with a cell-surface receptor termed receptor for AGEs (RAGE) elicits oxidative stress generation and subsequently evokes proliferative, angiogenic, and inflammatory reactions, thereby being involved in the development and progression of various types of cancers. These observations suggest that accumulation of AGEs and resultant activation of the RAGE signaling pathway could partly explain the increased risk of a variety of cancers in patients with diabetes or in elderly subjects. This article summarizes the pathological role of RAGE activation by AGEs and other ligands in tumor growth and metastasis and its therapeutic interventions for the life-threatening disorders.

Extracellular matrix is modulated in advanced glycation end products milieu via a RAGE receptor dependent pathway boosted by transforming growth factor‐β1 在晚期糖基化终末产物环境中细胞外基质通过RAGE受体依赖性途径调节，转化生长因子‐β1可以促进这种调节

Interstitial fibrosis is induced by imbalances in extracellular matrix homeostasis. Advanced glycation end products (AGEs) can bind and activate the receptor for AGEs (RAGE), which is involved in diabetic nephropathy. We set out to identify the role of AGEs in producing alterations leading to matrix hypertrophy and the pathway through which aminoguanidine, as well as anti-RAGE and anti-transforming growth factor (TGF)-β1 antibody treatments could prevent these modifications. Human embryonic kidney (HEK-293) cells were exposed to glycated bovine serum albumin (AGE-BSA) and co-treated with neutralizing antibodies or aminoguanidine. The effects on the transcriptional and translational levels of RAGE, TGF-β1 and collagen IV were evaluated, while metalloproteinase activity was assessed by gelatin zymography. AGE-BSA (200 μg/mL) upregulated RAGE's expression, while TGF-β1 synthesis and the formation of its bioactive form were increased in a dose-dependent manner by AGEs. AGE-BSA exposure increased both matrix metalloproteinase (MMP) activity and collagen IV synthesis, boosted by TGF-β1 upregulation. Aminoguanidine's effects revealed that small concentrations (10 μmol/L) enhance AGE-BSA effects, by increasing the expression of RAGE and TGF-β1, while higher concentrations (100 μmol/L) contribute to their downregulation. Although AGEs regulate RAGE and TGF-β1 by distinct pathways, RAGE activation leads to a further increase of TGF-β1 levels. MMP-2 activity seems to rely on TGF-β1, while MMP-9 was dependent on RAGE. These factors converge to control collagen IV turnover. Furthermore, although the antibody treatments might appear more efficient than AG in decreasing collagen IV levels, the cells compensate the RAGE and TGF-β1 blockade by increasing the mRNA expression of these proteins.

Possible role of methylglyoxal and glyoxalase in arthritis

OA (osteoarthritis) and RA (rheumatoid arthritis) lead to deterioration of the joints. Early OA is associated with loss of bone due to increased bone remodelling. A role for inflammation is thought to be integral to the pathology. RA is a chronic inflammatory disease of the synovium, a membrane lining the non-weight-bearing surfaces of the joint. The mainstay of RA diagnostic testing is for autoantibodies. Rheumatoid factor has been a primary diagnostic test; however, sensitivity is approximately 75%, but specificity is limited. Recently, detection of antibodies against cyclic citrullinated peptide, identified as a screening marker and marker of disease progression, has been proposed. Studies of glycation in arthritis have focused mostly on levels of AGEs (advanced glycation end-products), Nε-carboxymethyl-lysine and pentosidine. There was a weak correlation of skin and urinary pentosidine with joint damage in early-stage OA. RAGE (receptor for AGEs) is a cell-surface receptor in the synovial tissue of patients with OA and RA. The RAGE agonist S100A12 is increased in RA and OA. Activation of RAGE may decrease expression of Glo1 (glyoxalase I). Conflict between RAGE-activated inflammatory signalling and Nrf2 (nuclear factor-erythroid 2-related factor 2) regulation of basal and inducible expression of Glo1 may be involved. Thereby glyoxal- and methylglyoxal-derived AGEs may be increased in OA and RA. Further studies are now required to investigate the role of glyoxalase and dicarbonyl glycation in OA and RA for early-stage diagnosis and potential novel preventive therapy.

Targeting of Receptor for Advanced Glycation End Products Suppresses Cyst Growth in Polycystic Kidney Disease

Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited renal disorder. Although a myriad of research groups have attempted to identify a new therapeutic target for ADPKD, no drug has worked well in clinical trials. Our research group has focused on the receptor for advanced glycation end products (RAGE) gene as a novel target for ADPKD. This gene is involved in inflammation and cell proliferation. We have already confirmed that blocking RAGE function attenuates cyst growth in vitro. Based on this previous investigation, our group examined the effect of RAGE on cyst enlargement in vivo. PC2R mice, a severe ADPKD mouse model that we generated, were utilized. An adenovirus containing anti-RAGE shRNA was injected intravenously into this model. We observed that RAGE gene knockdown resulted in loss of kidney weight and volume. Additionally, the cystic area that originated from different nephron segments decreased in size because of down-regulation of the RAGE gene. Blood urea nitrogen and creatinine values tended to be lower after inhibiting RAGE. Based on these results, we confirmed that the RAGE gene could be an effective target for ADPKD treatment.

Reduction of Advanced-Glycation End Products Levels and Inhibition of RAGE Signaling Decreases Rat Vascular Calcification Induced by Diabetes

Advanced-glycation end products (AGEs) were recently implicated in vascular calcification, through a process mediated by RAGE (receptor for AGEs). Although a correlation between AGEs levels and vascular calcification was established, there is no evidence that reducing in vivo AGEs deposition or inhibiting AGEs-RAGE signaling pathways can decrease medial calcification. We evaluated the impact of inhibiting AGEs formation by pyridoxamine or elimination of AGEs by alagebrium on diabetic medial calcification. We also evaluated if the inhibition of AGEs-RAGE signaling pathways can prevent calcification. Rats were fed a high fat diet during 2 months before receiving a low dose of streptozotocin. Then, calcification was induced with warfarin. Pyridoxamine was administered at the beginning of warfarin treatment while alagebrium was administered 3 weeks after the beginning of warfarin treatment. Results demonstrate that AGEs inhibitors prevent the time-dependent accumulation of AGEs in femoral arteries of diabetic rats. This effect was accompanied by a reduced diabetes-accelerated calcification. Ex vivo experiments showed that N-methylpyridinium, an agonist of RAGE, induced calcification of diabetic femoral arteries, a process inhibited by antioxidants and different inhibitors of signaling pathways associated to RAGE activation. The physiological importance of oxidative stress was demonstrated by the reduction of femoral artery calcification in diabetic rats treated with apocynin, an inhibitor of reactive oxygen species production. We demonstrated that AGE inhibitors prevent or limit medial calcification. We also showed that diabetes-accelerated calcification is prevented by antioxidants. Thus, inhibiting the association of AGE-RAGE or the downstream signaling reduced medial calcification in diabetes.

Calgranulins May Contribute Vascular Protection In Atherogenesis

S100A8, S100A9 and S100A12 are considered proinflammatory mediators of atherosclerosis. Known as calgranulins, they are major components of neutrophils and are upregulated in macrophages and foam cells. They influence leukocyte recruitment, and may propagate inflammation by binding TLR4 and/or receptor for advanced glycation endproducts (RAGE). However, the receptors for calgranulins remain an enigma; we have no evidence for TLR4 or RAGE activation by S100A8 or S100A12. Moreover, gene regulation studies suggest antiinflammatory functions for S100A8 and emerging reports indicate pleiotropic roles. Unlike S100A9, S100A8 effectively scavenges oxidants generated by the myeloperoxidase system in vivo, forming novel thiol modifications. S100A8 is also readily S-nitrosylated, stabilizing nitric oxide and transporting it to hemoglobin. S100A8-SNO reduces leukocyte transmigration in the vasculature. S-glutathionylation of S100A9 modifies its effects on leukocyte adhesion. Both S100A8 forms inhibit mast cell activation, at least partially by scavenging reactive oxygen species required for signaling. Conversely, S100A12 activates and sequesters mast cells. However S100A12 suppresses proinflammatory cytokine induction by SAA-activated monocytes and macrophages, and inhibits matrix metalloprotease activity. We propose that the abundance and types of cells expressing calgranulins in particular microenvironments, their relative concentrations and post-translational modifications may have distinct functional outcomes, including those that are protective, at different stages of atherogenesis.

Dietary glycotoxins exacerbate progression of experimental fatty liver disease

Advanced glycation end-products (AGEs) levels are high in western diets and contribute to tissue injury via activation of RAGE (receptor for AGEs) and generation of reactive oxygen species (ROS). Here, we determined if high dietary AGE intake worsens progression of non-alcoholic fatty liver disease (NAFLD). Male Sprague Dawley rats were fed a methionine choline deficient (MCD) diet for 6 weeks before 6 weeks of a high AGE MCD diet through baking. They were compared with animals on MCD diet or a methionine choline replete (MCR) diet alone for 12 weeks. Hepatic ROS, triglycerides, biochemistry, picro-sirius morphometry, hepatic mRNA expression and immunohistochemistry were determined. Primary hepatic stellate cells (HSCs) from both MCR and MCD animals were exposed to AGEs. ROS, proliferation and mRNA expression were determined. The high AGE MCD diet increased hepatic AGE content and elevated triglycerides, NADPH dependent superoxide production, HNE adducts, steatosis, steatohepatitis (CD43, IL-6, TNF-α) and fibrosis (α-SMA, CTGF, COL1A, picrosirius) compared to MCD alone. In HSCs, AGEs significantly increased ROS production, bromodeoxyuridine proliferation and MCP-1, IL-6, α-SMA, and RAGE expression in HSCs from MCD but not MCR animals. These effects were abrogated by RAGE or NADPH oxidase blockade. In the MCD model of NAFLD, high dietary AGEs increases hepatic AGE content and exacerbates liver injury, inflammation, and liver fibrosis via oxidative stress and RAGE dependent profibrotic effects of AGEs on activated HSCs. This suggests that pharmacological and dietary strategies targeting the AGE/RAGE pathway could slow the progression of NAFLD.

The next generation of RAGE modulators: implications for soluble RAGE therapies in vascular inflammation

The next generation of RAGE modulators: implications for soluble RAGE therapies in vascular inflammation

Glycation exacerbates the neuronal toxicity of β-amyloid

Accumulation evidence shows that β-amyloid (Aβ) is a neurotoxic and accumulation of Aβ is responsible for the pathology of Alzheimer's disease (AD). However, it is currently not fully understood what makes Aβ toxic and accumulated. Previous studies demonstrate that Aβ is a suitable substrate for glycation, producing one form of the advanced glycation endproducts (AGEs). We speculated that Aβ-AGE formation may exacerbate the neurotoxicity. To explore whether the Aβ-AGE is more toxic than the authentic Aβ and to understand the molecular mechanisms, we synthesized glycated Aβ by incubating Aβ with methylglyoxal (MG) in vitro and identified the formation of glycated Aβ by fluorescence spectrophotometer. Then, we treated the primary hippocampal neurons cultured 8 days in vitro with Aβ-AGE or Aβ for 24 h. We observed that glycation exacerbated neurotoxicity of Aβ with upregulation of receptor for AGE (RAGE) and activation of glycogen synthase kinase-3 (GSK-3), whereas simultaneous application of RAGE antibody or GSK-3 inhibitor reversed the neuronal damages aggravated by glycated Aβ. Thereafter, we found that Aβ is also glycated with an age-dependent elevation of AGEs in Tg2576 mice, whereas inhibition of Aβ-AGE formation by subcutaneously infusion of aminoguanidine for 3 months significantly rescued the early cognitive deficit in mice. Our data reveal for the first time that the glycated Aβ is more toxic. We propose that the glycated Aβ with the altered secondary structure may be a more suitable ligand than Aβ for RAGE and subsequent activation of GSK-3 that can lead to cascade pathologies of AD, therefore glycated Aβ may be a new therapeutic target for AD.