Receptor For Advanced Glycation End-products Inhibition Research Articles

PM2.5 Exposure Aggravates Inflammatory Response and Mucus Production in 16HBE Cells through Inducing Oxidative Stress and RAGE Expression.

Particulate matter 2.5 (PM2.5)-induced oxidative stress has been extensively proposed as a pivotal event in lung diseases. Receptor for advanced glycation end-products (RAGE) is a receptor of pro-inflammatory ligands that has been supported to be implied in the progression of multiple lung diseases. This study attempts to delineate the specific effects of PM2.5 on human bronchial epithelial 16HBE cells in vitro and figure out whether PM2.5 functions via mediating oxidative stress and RAGE. In PM2.5-challenged 16HBE cells, MTT assay detected cell viability. ELISA estimated inflammatory levels. Flow cytometry analysis measured ROS activity and related assay kits examined oxidative stress levels. Western blot tested nuclear factor E2-related factor 2 (Nrf2), RAGE, β-catenin, and mucin 5AC (MUC5AC) expression. Immunofluorescence staining evaluated nuclear translocation of β-catenin. It was noticed that PM2.5 exposure exacerbated inflammatory response, oxidative stress, and mucus production. Additionally, PM2.5 elevated RAGE expression while declined Nrf2 expression as well as stimulated the nuclear translocation of β-catenin. Furthermore, RAGE inhibition or nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor VAS2870 mitigated inflammatory response, oxidative stress, and mucus generation in PM2.5-exposed 16HBE cells. In addition, RAGE inhibition or VAS2870 raised Nrf2 expression, reduced RAGE expression, and hampered β-catenin nuclear translocation. Briefly, PM2.5 might act as a leading driver of inflammatory response and mucus production in lung injury, the mechanism of which might be related to the activation of oxidative stress and the up-regulation of RAGE.

S100b treatment overcomes RAGE signaling deficits in myoblasts on advanced glycation end-product cross-linked collagen and promotes myogenic differentiation.

Advanced glycation end-products (AGEs) stochastically accrue in skeletal muscle and on collagen over an individual's lifespan, stiffening the muscle and modifying the stem cell (MuSC) microenvironment while promoting proinflammatory, antiregenerative signaling via the receptor for advanced glycation end-products (RAGEs). In the present study, a novel in vitro model was developed of this phenomenon by cross linking a 3-D collagen scaffold with AGEs and investigating how myoblasts responded to such an environment. Briefly, collagen scaffolds were incubated with d-ribose (0, 25, 40, 100, or 250 mM) for 5 days at 37°C. C2C12 immortalized mouse myoblasts were grown on the scaffolds for 6 days in growth conditions for proliferation, and 12 days for differentiation and fusion. Human primary myoblasts were also used to confirm the C2C12 data. AGEs aberrantly extended the DNA production stage of C2C12s (but not in human primary myoblasts) which is known to delay differentiation in myogenesis, and this effect was prevented by RAGE inhibition. Furthermore, the differentiation and fusion of myoblasts were disrupted by AGEs, which were associated with reductions in integrins and suppression of RAGE. The addition of S100b (RAGE agonist) recovered the differentiation and fusion of myoblasts, and the addition of RAGE inhibitors (FPS-ZM1 and Azeliragon) inhibited the differentiation and fusion of myoblasts. Our results provide novel insights into the role of the AGE-RAGE axis in skeletal muscle aging, and future work is warranted on the potential application of S100b as a proregenerative factor in aged skeletal muscle.NEW & NOTEWORTHY Collagen cross-linked by advanced glycation end-products (AGEs) induced myoblast proliferation but prevented differentiation, myotube formation, and RAGE upregulation. RAGE inhibition occluded AGE-induced myoblast proliferation, while the delivery of S100b, a RAGE ligand, recovered fusion deficits.

RAGE inhibitor TTP488 (Azeliragon) suppresses metastasis in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is a highly aggressive and metastatic cancer subtype, which is generally untreatable once it metastasizes. We hypothesized that interfering with the Receptor for Advanced Glycation End-products (RAGE) signaling with the small molecule RAGE inhibitors (TTP488/Azeliragon and FPS-ZM1) would impair TNBC metastasis and impair fundamental mechanisms underlying tumor progression and metastasis. Both TTP488 and FPS-ZM1 impaired spontaneous and experimental metastasis of TNBC models, with TTP488 reducing metastasis to a greater degree than FPS-ZM1. Transcriptomic analysis of primary xenograft tumor and metastatic tissue revealed high concordance in gene and protein changes with both drugs, with TTP488 showing greater potency against metastatic driver pathways. Phenotypic validation of transcriptomic analysis by functional cell assays revealed that RAGE inhibition impaired TNBC cell adhesion to multiple extracellular matrix proteins (including collagens, laminins, and fibronectin), migration, and invasion. Neither RAGE inhibitor impaired cellular viability, proliferation, or cell cycle in vitro. Proteomic analysis of serum from tumor-bearing mice revealed RAGE inhibition affected metastatic driver mechanisms, including multiple cytokines and growth factors. Further mechanistic studies by phospho-proteomic analysis of tumors revealed RAGE inhibition led to decreased signaling through critical BC metastatic driver mechanisms, including Pyk2, STAT3, and Akt. These results show that TTP488 impairs metastasis of TNBC and further clarifies the signaling and cellular mechanisms through which RAGE mediates metastasis. Importantly, as TTP488 displays a favorable safety profile in human studies, our study provides the rationale for evaluating TTP488 in clinical trials to treat or prevent metastatic TNBC.

RAGE contributes to allergen driven severe neutrophilic airway inflammation via NLRP3 inflammasome activation in mice.

Asthma is a major public healthcare burden, affecting over 300 million people worldwide. While there has been great progress in the treatment of asthma, subsets of patients who present with airway neutrophilia, often have more severe disease, and tend to be resistant to conventional corticosteroid treatments. The receptor for advanced glycation endproducts (RAGE) plays a central role in the pathogenesis of eosinophilic asthma, however, it's role in neutrophilic asthma remains largely uninvestigated. A mouse model of severe steroid resistant neutrophilic airway disease (SSRNAD) using the common fungal allergen Alternaria alternata (AA) was employed to evaluate the effects of genetic ablation of RAGE and pharmacological inhibition of the NLRP3 inflammasome on neutrophilic airway inflammation. AA exposure induced robust neutrophil-dominant airway inflammation and increased BALF levels of Th1/Th17 cytokines in wild-type mice, which was significantly reduced in RAGE-/- mice. Serum levels of IgE and IgG1 were increased similarly in both wild-type and RAGE-/- mice. Pharmacological inhibition of NLRP3 blocked the effects of AA exposure and NLRP3 inflammasome activation was RAGE-dependent. Neutrophil extracellular traps were elevated in the BALF of wild-type but not RAGE-/- mice and an atypical population of SiglecF+ neutrophils were identified in the BALF. Lastly, time-course studies found that RAGE expression promoted sustained neutrophil accumulation in the BALF of mice in response to AA.

A Review on Inhibition of RAGE via Marine Sources, Herbal Sources and Food Substances

Receptor for Advanced Glycation End-products (RAGE), also known as AGER,is a 35 kilodalton transmembrane receptor of the Immunoglobulin super family. Its name is mainly due to its ability to bind to advanced glycation end products (AGE), including glycoproteins and glycans which have been modified non-enzymatically through the Maillard reaction. RAGE is referred to as a Pattern Recognition Receptor. Studies have determined the contribution of protein glycation to disease-states and have mainly aimed at the harmful effects and mechanisms of these glycotoxins. Thus, the development and testing of AGE inhibitors, especially natural anti-AGE formulations, i.e. RAGE inhibitors without any side effects, may provide a therapeutic approach. In particular, the pursuit of RAGE inhibitors using in vitro and in vivo models identifies naturally occurring compounds for preventing glycation. This leads to inhibition of RAGE. Synthetic compounds also can inhibit the RAGE. Available data suggests that natural and synthetic compounds which have certain chemical constituents, may attenuate glycation, and can lead to RAGE inhibition via Natural as well as synthetic Sources.

Receptor for Advanced Glycation End-Products Promotes Activation of Alveolar Macrophages through the NLRP3 Inflammasome/TXNIP Axis in Acute Lung Injury.

The roles of thioredoxin-interacting protein (TXNIP) and receptor for advanced glycation end-products (RAGE)-dependent mechanisms of NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome-driven macrophage activation during acute lung injury are underinvestigated. Cultured THP-1 macrophages were treated with a RAGE agonist (S100A12), with or without a RAGE antagonist; cytokine release and intracytoplasmic production of reactive oxygen species (ROS) were assessed in response to small interfering RNA knockdowns of TXNIP and NLRP3. Lung expressions of TXNIP and NLRP3 and alveolar levels of IL-1β and S100A12 were measured in mice after acid-induced lung injury, with or without administration of RAGE inhibitors. Alveolar macrophages from patients with acute respiratory distress syndrome and from mechanically ventilated controls were analyzed using fluorescence-activated cell sorting. In vitro, RAGE promoted cytokine release and ROS production in macrophages and upregulated NLRP3 and TXNIP mRNA expression in response to S100A12. TXNIP inhibition downregulated NLRP3 gene expression and RAGE-mediated release of IL-1β by macrophages in vitro. In vivo, RAGE, NLRP3 and TXNIP lung expressions were upregulated during experimental acute lung injury, a phenomenon being reversed by RAGE inhibition. The numbers of cells expressing RAGE, NLRP3 and TXNIP among a specific subpopulation of CD16+CD14+CD206- (“pro-inflammatory”) alveolar macrophages were higher in patients with lung injury. This study provides a novel proof-of-concept of complex RAGE–TXNIP–NLRP3 interactions during macrophage activation in acute lung injury.

LL-37 and HMGB1 induce alveolar damage and reduce lung tissue regeneration via RAGE.

The receptor for advanced glycation end-products (RAGE) has been implicated in the pathophysiology of chronic obstructive pulmonary disease (COPD). However, it is still unknown whether RAGE directly contributes to alveolar epithelial damage and abnormal repair responses. We hypothesize that RAGE activation not only induces lung tissue damage but also hampers alveolar epithelial repair responses. The effects of the RAGE ligands LL-37 and HMGB1 were examined on airway inflammation and alveolar tissue damage in wild-type and RAGE-deficient mice and on lung damage and repair responses using murine precision cut lung slices (PCLS) and organoids. In addition, their effects were studied on the repair response of human alveolar epithelial A549 cells, using siRNA knockdown of RAGE and treatment with the RAGE inhibitor FPS-ZM1. We observed that intranasal installation of LL-37 and HMGB1 induces RAGE-dependent inflammation and severe alveolar tissue damage in mice within 6 h, with stronger effects in a mouse strain susceptible for emphysema compared with a nonsusceptible strain. In PCLS, RAGE inhibition reduced the recovery from elastase-induced alveolar tissue damage. In organoids, RAGE ligands reduced the organoid-forming efficiency and epithelial differentiation into pneumocyte-organoids. Finally, in A549 cells, we confirmed the role of RAGE in impaired repair responses upon exposure to LL-37. Together, our data indicate that activation of RAGE by its ligands LL-37 and HMGB1 induces acute lung tissue damage and that this impedes alveolar epithelial repair, illustrating the therapeutic potential of RAGE inhibitors for lung tissue repair in emphysema.

Involvement of receptor for advanced glycation end products in microgravity-induced skeletal muscle atrophy in mice

The accumulation of advanced glycation end-products (AGEs) may be involved in the mechanism of skeletal muscle atrophy. However, the involvement of the receptor for AGEs (RAGE) axis in microgravity-induced skeletal muscle atrophy has not been investigated. Therefore, the purpose of the present study was to investigate the effect of RAGE inhibition on microgravity-induced skeletal muscle atrophy and the related molecular responses. Male C57BL/6NCr mice subjected to a 1-week hindlimb suspension lead to muscle atrophy in soleus and plantaris but not extensor digitorum longus muscle, accompanied by increases in RAGE expression. However, treatment with a RAGE antagonist (FPS-ZM1, intraperitoneal, 1 mg/kg/day) during hindlimb suspension ameliorated the atrophic responses in soleus muscle. Further, muscle mass inversely correlated with the accumulation of AGEs (methylglyoxal-modified proteins and Nε-(carboxymethyl) lysine-modified proteins) in soleus muscle. The expression of proinflammatory cytokines, tumor necrosis factor-α, interleukin-1β, and interleukin-6 in soleus muscle was enhanced in response to hindlimb suspension, but these changes were attenuated by FPS-ZM1 treatment. Protein ubiquitination and ubiquitin E3 ligase (muscle RING finger 1) expression in soleus muscle were elevated following hindlimb suspension, and these increments were suppressed by FPS-ZM1 treatment. Our findings indicate that the AGE-RAGE axis is upregulated in unloaded atrophied skeletal muscle, and that RAGE inhibition ameliorates microgravity-induced skeletal muscle atrophy by reducing proinflammatory cytokine expression and ubiquitin-proteasome system activation.

The receptor for advanced glycation end-products enhances lung epithelial wound repair: An in vitro study

Re-epithelialization of the alveolar surface is a key process of lung alveolar epithelial barrier repair after acute lung injury. The receptor for advanced glycation end-products (RAGE) pathway plays key roles in lung homeostasis, and its involvement in wound repair has been already reported in human bronchial epithelial cells. However, its effects on lung alveolar epithelial repair after injury remain unknown. We investigated whether RAGE stimulation with its ligands high–mobility group box 1 protein (HMGB1) or advanced glycation end-products (AGEs), alone or associated with RAGE inhibition using RAGE antagonist peptide, affects in vitro wound healing in human alveolar epithelial A549 cells. We further asked whether these effects could be associated with changes in cell proliferation and migration. We found that treatment of A549 cells with HMGB1 or AGEs promotes RAGE-dependent wound healing after a scratch assay. In addition, both RAGE ligands increased cell proliferation in a RAGE-dependent manner. Treatment with HMGB1 increased migration of alveolar epithelial cells at 12 h, independently of RAGE, whereas AGEs stimulated migration as measured 48 h after injury in a RAGE-dependent manner. Taken together, these results suggest that RAGE pathway is involved in lung alveolar epithelial wound repair, possibly through enhanced cell migration and proliferation.

Abstract P3-01-11: Targeting RAGE inhibits breast cancer invasion and metastasis

Abstract Background: Breast cancer (BC) is treatable with early detection, but once metastasis occurs, there is no cure. Therefore, it is of upmost importance to find targetable biomarkers that are involved in modulating the multi-step metastatic process. The Receptor for Advanced Glycation Endproducts (RAGE) and its ligands are an inflammatory pathway that are involved in modulating breast cancer progression and metastasis. We recently demonstrated that genetic or pharmacological interruption of RAGE signaling affected tumor progression and metastasis. However, no studies have dissected the role of RAGE in tumor growth from the metastatic cascade. Here, we show for the first time in multiple metastatic breast cancer models that targeting RAGE impairs BC metastasis. Methods: We tested the anti-metastatic effect of RAGE in vitro using the RAGE inhibitor FPS-ZM1 or RAGE shRNA knockdown in cell invasion, proliferation, migration and sphere formation assays (with 4T1, Py8119 and E0771 mouse BC cells). For in vivo assays we used orthotopic BC models (4T1/BALB/c and E0771 & Py8119/C57BL6) and experimental metastasis assays (tail vein injection of 4T1/BALB/c and Py8119/C57BL6). To target RAGE, we used genetic (shRNA in 4T1 cells and RAGE knockout in C57BL6 mice) and pharmacological approaches (I.P. injection of FPS-ZM1). To investigate the synergistic effects of RAGE inhibition on progression and metastasis, we tested combination therapy of low-dose doxorubicin and FPS-ZM1. Results: Inhibition of RAGE with FPS-ZM1 (1-5uM) impaired tumor cell invasion of 4T1, E0771 and Py8119 cells. Similarly, in spheroid assays, treatment of cells with FPS-ZM1 resulted in fewer and smaller colonies. However, FPS-ZM1 treatment did not affect cell proliferation. In vivo studies revealed that FPS-ZM1 (1mg/kg) has a modest effect on tumor growth in 4T1/BALB/c injected mice but displayed a dramatic inhibitory effect on metastasis to the lungs. In experimental metastasis assays, tail-vein injection of 4T1 cells in BALB/c mice demonstrated that FPS-ZM1 treatment strongly impaired metastatic disease in mice compared to controls. To dissect the genetic role of RAGE in the tumor versus host, we test the effect of RAGE knockdown in 4T1 cells in experimental metastasis assays. RAGE shRNA impaired metastasis to the lungs, albeit to a lower degree seen with the RAGE inhibitor. To test the role of the host, we injected Py8119 cells into wild-type and RAGE knockout (RKO) mice. RKO mice displayed fewer metastatic burden compared to wild-type mice. Finally, in our combination treatment experiments, treatment of 4T1-injected BALB/c mice with Doxorubicin and FPS-ZM1 (alone and in combination), demonstrated that drug combination was more effective in inhibiting lung metastasis than either reagent alone. We are currently assessing how RAGE mechanistically drives these metastatic changes. Conclusion: Our data strongly suggests RAGE plays an important role in breast cancer metastasis, with less of an effect on tumor growth. Ongoing studies in our lab are testing which stage of the metastatic cascade RAGE is involved in, and the underlying mechanisms driving these processes. In conclusion, the use of RAGE inhibitors could represent a novel therapeutic approach for metastatic breast cancer. Citation Format: Gyong Ha Hwang, Melinda Magna, Barbara Mera, Toni Yeasky, Taekyoung Kwak, Lucas Outcault, Masaru Takabatake, Thuy-Mai Le, Marc E. Lippman, Barry I. Hudson. Targeting RAGE inhibits breast cancer invasion and metastasis [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-01-11.

Inhibition of Receptor for Advanced Glycation End Products as New Promising Strategy Treatment in Diabetic Retinopathy.

BACKGROUND:Extensive intracellular and extracellular formation of advanced glycation end-products (AGEs) is considered a causative factor for vascular injury triggered by hyperglycemia in diabetes. The hyperglycemia will cause accumulation of AGEs, damage to pericytes, nerve growth factor (NGF), glial acid fibrillary protein (GFAP) and increase in vascular endothelial growth factor (VEGF).AIM:This study aimed to assess the efficacy of RAGE inhibition in suppressing the development and progression of diabetic retinopathy through modulation of the inflammatory pathway involving NGF, GFAP, and VEGF.METHODS:The design was in vivo experimental study. Thirty white rats were induced with Alloxan monohydrate. Rats were divided into 5 groups, normal, negative control, groups with an anti-RAGE dose of 1 μg/uL, the dose of 10 μg/uL and 100 μg/uL. After 4 weeks of treatment, HbA1c, NGF, and GFAP levels were measured using ELISA. Quantification of VEGF expression was done using the ImageJ® application. Data was expressed with mean ± SD. Independent T-test with ANOVA and Tukey’s post hoc was done.RESULTS:RAGE inhibitors yielded a significant decrease in blood glucose and HbA1c levels. VEGF and RAGE expression were reduced in anti-RAGE groups in various doses. Inhibition of RAGE reduced the damage of retinal pericytes, by reducing GFAP and increasing NGF, and reduced the formation of new blood vessels, by decreasing VEGF expression, in diabetic retinopathy.CONCLUSION:Inhibition of receptor for advanced glycation end-products (RAGE) was effective in suppressing the development and progression of diabetic retinopathy.

Inhibition of the Receptor for Advanced Glycation End-Products in Acute Respiratory Distress Syndrome: A Randomised Laboratory Trial in Piglets

The receptor for advanced glycation end-products (RAGE) modulates the pathogenesis of acute respiratory distress syndrome (ARDS). RAGE inhibition attenuated lung injury and restored alveolar fluid clearance (AFC) in a mouse model of ARDS. However, clinical translation will require assessment of this strategy in larger animals. Forty-eight anaesthetised Landrace piglets were randomised into a control group and three treatment groups. Animals allocated to treatment groups underwent orotracheal instillation of hydrochloric acid (i) alone; (ii) in combination with intravenous administration of a RAGE antagonist peptide (RAP), or (iii) recombinant soluble (s)RAGE. The primary outcome was net AFC at 4 h. Arterial oxygenation was assessed hourly and alveolar-capillary permeability, alveolar inflammation and lung histology were assessed at 4 h. Treatment with either RAP or sRAGE improved net AFC (median [interquartile range], 21.2 [18.8–21.7] and 19.5 [17.1–21.5] %/h, respectively, versus 12.6 [3.2–18.8] %/h in injured, untreated controls), oxygenation and decreased alveolar inflammation and histological evidence of tissue injury after ARDS. These findings suggest that RAGE inhibition restored AFC and attenuated lung injury in a piglet model of acid-induced ARDS.

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.

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.

Inhibition of the receptor for advanced glycation end-products (RAGE) protects from secondhand smoke (SHS)-induced intrauterine growth restriction IUGR in mice.

Intrauterine growth restriction (IUGR) is a disease affecting 10% of all pregnancies. IUGR is associated with maternal, fetal, or placental abnormalities. Studies investigating the effects of secondhand smoke (SHS) exposure and IUGR are limited. The receptor for advanced glycation end-products (RAGE) is a pro-inflammatory transmembrane receptor increased by SHS in the placenta. We tested the hypothesis that inhibition of RAGE during SHS exposure protects from smoke-induced IUGR. C57BL/6 mice were exposed to SHS or SHS+semi-synthetic glycosaminoglycan ethers (SAGEs) known to inhibit RAGE signaling. Trophoblast cells were treated with cigarette smoke extract (CSE) with or without SAGEs in order to address the effects of RAGE inhibition during trophoblast invasion in vitro. SHS-treated mice demonstrated a significant reduction in fetal weight (7.35-fold, P≤0.0001) and placental weight (1.13-fold, P≤0.0001) compared with controls. Mice co-treated with SHS and SAGEs were protected from SHS-induced fetal weights decreases. SHS treatment of C57BL/6 mice activated placental extracellular signal-regulated kinase (ERK) (3.0-fold, P≤0.05), JNK (2.4-fold, P≤0.05) and p38 (2.1-fold, P≤0.05) and the expression of inflammatory mediators including TNF-α (1.34-fold, P≤0.05) and IL-1β (1.03-fold, P≤0.05). SHS-mediated activation of these molecules was reduced to basal levels when SAGE was co-administered. Invasion of trophoblast cells decreased 92% (P<0.002) when treated with CSE and CSE-mediated invasion was completely reversed by SAGEs. We conclude that RAGE inhibition protects against fetal weight loss during SHS-induced IUGR. These studies provide insight into tobacco-mediated IUGR development and clarify avenues that may be helpful in the alleviation of placental complications.

Targeted inhibition of RAGE in substantia nigra of rats blocks 6-OHDA\u2013induced dopaminergic denervation

The receptor for advanced glycation endproducts (RAGE) is a pattern-recognition receptor associated with inflammation in most cell types. RAGE up-regulates the expression of proinflammatory mediators and its own expression via activation of NF-kB. Recent works have proposed a role for RAGE in Parkinson’s disease (PD). In this study, we used the multimodal blocker of RAGE FPS-ZM1, which has become available recently, to selectively inhibit RAGE in the substantia nigra (SN) of rats intracranially injected with 6-hydroxydopamine (6-OHDA). FPS-ZM1 (40 μg per rat), injected concomitantly with 6-OHDA (10 μg per rat) into the SN, inhibited the increase in RAGE, activation of ERK1/2, Src and nuclear translocation of NF-kB p65 subunit in the SN. RAGE inhibition blocked glial fibrillary acidic protein and Iba-1 upregulation as well as associated astrocyte and microglia activation. Circulating cytokines in serum and CSF were also decreased by FPS-ZM1 injection. The loss of tyrosine hydroxylase and NeuN-positive neurons was significantly inhibited by RAGE blocking. Finally, FPS-ZM1 attenuated locomotory and exploratory deficits induced by 6-OHDA. Our results demonstrate that RAGE is an essential component in the neuroinflammation and dopaminergic denervation induced by 6-OHDA in the SN. Selective inhibition of RAGE may offer perspectives for therapeutic approaches.

RAGE inhibition reduces acute lung injury in mice

The receptor for advanced glycation end-products (RAGE) is involved in inflammatory response during acute respiratory distress syndrome (ARDS). Growing body of evidence support strategies of RAGE inhibition in experimental lung injury, but its modalities and effects remain underinvestigated. Anesthetised C57BL/6JRj mice were divided in four groups; three of them underwent orotracheal instillation of acid and were treated with anti-RAGE monoclonal antibody (mAb) or recombinant soluble RAGE (sRAGE), acting as a decoy receptor. The fourth group served as a control. Lung injury was assessed by the analysis of blood gases, alveolar permeability, histology, AFC, and cytokines. Lung expression and distribution epithelial channels ENaC, Na,K-ATPase, and aquaporin (AQP)−5 were assessed. Treatment with either anti-RAGE mAb or sRAGE improved lung injury, arterial oxygenation and decreased alveolar inflammation in acid-injured animals. Anti-RAGE therapies were associated with restored AFC and increased lung expression of AQP-5 in alveolar cell. Blocking RAGE had potential therapeutic effects in a translational mouse model of ARDS, possibly through a decrease in alveolar type 1 epithelial cell injury as shown by restored AFC and lung AQP-5 expression. Further mechanistic studies are warranted to describe intracellular pathways that may control such effects of RAGE on lung epithelial injury and repair.

Anti-RAGE antibody selectively blocks acute systemic inflammatory responses to LPS in serum, liver, CSF and striatum

Systemic inflammation induces transient or permanent dysfunction in the brain by exposing it to soluble inflammatory mediators. The receptor for advanced glycation endproducts (RAGE) binds to distinct ligands mediating and increasing inflammatory processes. In this study we used an LPS-induced systemic inflammation model in rats to investigate the effect of blocking RAGE in serum, liver, cerebrospinal fluid (CSF) and brain (striatum, prefrontal cortex, ventral tegmental area and substantia nigra). Intraperitoneal injection of RAGE antibody (50μg/kg) was followed after 1h by a single LPS (5mg/kg) intraperitoneal injection. Twenty-four hours later, tissues were isolated for analysis. RAGE antibody reduced LPS-induced inflammatory effects in both serum and liver; the levels of proinflammatory cytokines (TNF-α, IL-1β) were decreased and the phosphorylation/activation of RAGE downstream targets (ERK1/2, IκB and p65) in liver were significantly attenuated. RAGE antibody prevented LPS-induced effects on TNF-α and IL-1β in CSF. In striatum, RAGE antibody inhibited increases in IL-1β, Iba-1, GFAP, phospho-ERK1/2 and phospho-tau (ser202), as well as the decrease in synaptophysin levels. These effects were caused by systemic RAGE inhibition, as RAGE antibody did not cross the blood-brain barrier. RAGE antibody also prevented striatal lipoperoxidation and activation of mitochondrial complex II. In conclusion, blockade of RAGE is able to inhibit inflammatory responses induced by LPS in serum, liver, CSF and brain.

Receptor for advanced glycation end-products regulates lung fluid balance via protein kinase C-gp91(phox) signaling to epithelial sodium channels.

The receptor for advanced glycation end-products (RAGE), a multiligand member of the Ig family, may play a crucial role in the regulation of lung fluid balance. We quantified soluble RAGE (sRAGE), a decoy isoform, and advanced glycation end-products (AGEs) from the bronchoalveolar lavage fluid of smokers and nonsmokers, and tested the hypothesis that AGEs regulate lung fluid balance through protein kinase C (PKC)-gp91(phox) signaling to the epithelial sodium channel (ENaC). Human bronchoalveolar lavage samples from smokers showed increased AGEs (9.02 ± 3.03 μg versus 2.48 ± 0.53 μg), lower sRAGE (1,205 ± 292 pg/ml versus 1,910 ± 263 pg/ml), and lower volume(s) of epithelial lining fluid (97 ± 14 ml versus 133 ± 17 ml). sRAGE levels did not predict ELF volumes in nonsmokers; however, in smokers, higher volumes of ELF were predicted with higher levels of sRAGE. Single-channel patch clamp analysis of rat alveolar epithelial type 1 cells showed that AGEs increased ENaC activity measured as the product of the number of channels (N) and the open probability (Po) (NPo) from 0.19 ± 0.08 to 0.83 ± 0.22 (P = 0.017) and the subsequent addition of 4-hydroxy-2, 2, 6, 6-tetramethylpiperidine-N-oxyl decreased ENaC NPo to 0.15 ± 0.07 (P = 0.01). In type 2 cells, human AGEs increased ENaC NPo from 0.12 ± 0.05 to 0.53 ± 0.16 (P = 0.025) and the addition of 4-hydroxy-2, 2, 6, 6-tetramethylpiperidine-N-oxyl decreased ENaC NPo to 0.10 ± 0.03 (P = 0.013). Using molecular and biochemical techniques, we observed that inhibition of RAGE and PKC activity attenuated AGE-induced activation of ENaC. AGEs induced phosphorylation of p47(phox) and increased gp91(phox)-dependent reactive oxygen species production, a response that was abrogated with RAGE or PKC inhibition. Finally, tracheal instillation of AGEs promoted clearance of lung fluid, whereas concomitant inhibition of RAGE, PKC, and gp91(phox) abrogated the response.

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.