Levels Of Receptor For Advanced Glycation End-products Research Articles

Role of Blood-Brain Barrier in Alzheimer's Disease.

The blood-brain barrier (BBB) is involved in the pathogenesis of Alzheimer's disease (AD). BBB is a highly selective semipermeable structural and chemical barrier which ensures a stable internal environment of the brain and prevents foreign objects invading the brain tissue. BBB dysfunction induces the failure of Aβ transport from brain to the peripheral circulation across the BBB. Especially, decreased levels of LRP-1 (low density lipoprotein receptor-related protein 1) and increased levels of RAGE (receptor for advanced glycation endproducts) at the BBB can cause the failure of Aβ transport. The pathogenesis of AD is related to the BBB structural components, including pericytes, astrocytes, vascular endothelial cells, and tight junctions. BBB dysfunction will trigger neuroinflammation and oxidative stress, then enhance the activity of β-secretase and γ-secretase, and finally promote Aβ generation. A progressive accumulation of Aβ in brain and BBB dysfunction may become a feedback loop that gives rise to cognitive impairment and the onset of dementia. The correlation between BBB dysfunction and tau pathology has been well-reported. Therefore, regulating BBB function may be a new therapeutic target for treating AD.

Influence of high mobility group box 1 (HMGB1) derived from SCC7 cells on mouse normal tongue muscle fibers

ObjectiveHigh mobility group box 1 (HMGB1) is present in the nucleus of all normal cells and known for its participation in the maintenance of homeostasis. But recent studies suggested an association with the invasion and metastasis of cancer. Therefore, we aimed to clarify how HMGB1 released from cancer cells affects mice tongue muscle fibers. MethodsIn this study, we conducted immunohistochemical investigation of the localization of HMGB1 and the receptor for advanced glycation endproducts (RAGE) in the carcinoma and adjacent muscle tissues of the consecutive serial frontal section of mice tongues, into which SCC7 cells (isolated from a murine squamous cell carcinoma) had been implanted. The amounts of HMGB1 and RAGE mRNAs expressed were also examined. ResultsAt first, observation of hematoxylin/eosin-stained sections showed muscle fibers that had become reduced in size in the anterior and middle tongue from the SCC7 group. Immunohistochemical staining revealed that HMGB1 and RAGE were expressed not only in the carcinoma but also in the muscle fibers peripheral of the carcinoma and distal to the carcinoma. As with the immunohistochemical staining, mRNA expression levels of HMGB1 and RAGE were also expressed in the carcinoma and peripheral/distal muscle fibers. ConclusionThese results indicated the HMGB1 protein released from the carcinoma had reduced the size of the muscle fibers, through the RAGE. Furthermore, the possibility that this promotes cancer invasion through the formation of a clearance gap in high density skeletal muscle tissue was suggested.

Detection of the receptor for advanced glycation endproducts in neuronally-derived exosomes in plasma

Exosomes are nanovesicles that participate in cell-to-cell communication and are secreted by a variety of cells including neurons. Recent studies suggest that neuronally-derived exosomes are detectable in plasma and that their contents likely reflect expression of various biomarkers in brain tissues. The receptor for advanced glycation endproducts (RAGE) has been implicated in the pathophysiology of Alzheimer's disease (AD) and is increased in brain regions affected by AD. The goal of our project was to determine whether RAGE is present in plasma exosomes, and specifically exosomes derived from neurons. Exosomes were isolated from plasma samples (n = 8) by precipitation (ExoQuick) and ultracentrifugation methods. Neuronally-derived exosomes were isolated using a biotin-tagged L1 Cell Adhesion Molecule (L1CAM) specific antibody and streptavidin-tagged agarose resin. RAGE expression was measured by Western blots and ELISA. Western Blotting showed that RAGE is present in L1CAM-positive exosomes isolated using both methods. Mean (SD) exosomal RAGE levels were 164 (60) pg/ml by ExoQuick and were highly correlated with plasma sRAGE levels (r = 0.87, p = 0.005), which were approximately 7.5-fold higher than exosomal levels. Weak to moderate correlations were found between exosomal RAGE and age, BMI, and cognitive function. These results show for the first time that RAGE is present in neuronally-derived plasma exosomes, and suggest that exosomal RAGE may be a novel biomarker that reflects pathophysiological processes in the brain.

High glucose promotes tumor cell proliferation and migration in lung adenocarcinoma via the RAGE‑NOXs pathway.

Over the past few decades, it has been demonstrated that hyperglycemia can promote lung carcinoma growth, potentially through significantly increased glucose metabolism; however, the underlying mechanism remains to be fully elucidated. In the present study, treatment with a high concentration of glucose (HG) significantly promoted the proliferation and migration of A549 cells. Receptor for advanced glycation end‑products (RAGE) has previously been demonstrated to be associated with diabetes mellitus and oxidative stress, and nicotinamide adenine dinucleotide phosphate oxidases (NOXs) are considered to be initiating factors of oxidative stress. Therefore, an MTT assay, wound‑healing assay, quantitative polymerase chain reaction and western blotting assays were used to analyze the RAGE‑NOX‑4 pathway and to determine its potential involvement in glycometabolism‑associated tumorigenesis. The present study demonstrated that HG could increase the protein expression of RAGE and NOX‑4, whereas the inhibitor of RAGE (anti‑RAGE antibody) could suppress this effect. Futhermore, the inhibitor of NOX [diphenyl iodonium chloride (DPI)] could reduce the protein expression of RAGE and NOX‑4. Furthermore, inhibition of RAGE led to the downregulation of vascular endothelial growth factor (VEGF) and hypoxia‑inducible factor‑1α (HIF‑1α), thus suggesting that HG may influence angiogenesis and tumor metabolism via the RAGE‑NOXs pathway. The present study also demonstrated that the RAGE‑blocking antibody downregulated NOX‑4 and subsequently reduced the production of downstream inflammatory factors, whereas DPI did not affect the mRNA expression of RAGE but it did reduce the protein level of RAGE and then attenuate the inflammatory response. These results indicated that inhibition of RAGE or NOXs may promote the reduced expression of VEGF and HIF‑1α, and NOXs may be downstream targets of RAGE, thus indicating a HG‑RAGE‑NOXs‑VEGF/HIF‑1α association. Furthermore, the results indicated that HG may serve a role in the development of lung adenocarcinoma, mediated by the RAGE‑oxidative stress pathway; therefore, the regulation of this glucose‑associated pathway may be a promising novel direction for oncotherapy. However, while certain antidiabetic agents have been verified to exert inhibitory effects on tumor growth, they can also have long‑term adverse effects on the body, which may limit the value of these drugs as anticancer treatments. In conclusion, the present study suggested a novel attempt to suppress glucose‑induced tumor growth using a RAGE inhibitor such as soluble RAGE while avoiding the risk of glucose fluctuation.

Multimodal imaging of the receptor for advanced glycation end-products with molecularly targeted nanoparticles.

The receptor for advanced glycation end-products (RAGE) is central to multiple disease states, including diabetes-related conditions such as peripheral arterial disease (PAD). Despite RAGE's importance in these pathologies, there remains a need for a molecular imaging agent that can accurately assess RAGE levels in vivo. Therefore, we have developed a multimodal nanoparticle-based imaging agent targeted at RAGE with the well-characterized RAGE ligand, carboxymethyllysine (CML)-modified human serum albumin (HSA).Methods: A multimodal tracer (64Cu-Rho-G4-CML) was developed using a generation-4 (G4) polyamidoamine (PAMAM) dendrimer, conjugated with both rhodamine and copper-64 (64Cu) chelator (NOTA) for optical and PET imaging, respectively. First, 64Cu-Rho-G4-CML and its non-targeted analogue (64Cu-Rho-G4-HSA) were evaluated chemically using techniques such as dynamic light scattering (DLS), electron microscopy and nuclear magnetic resonance (NMR). The tracers' binding capabilities were examined at the cellular level and optimized using live and fixed HUVEC cells grown in 5.5-30 mM glucose, followed by in vivo PET-CT imaging, where the probes' kinetics, biodistribution, and RAGE targeting properties were examined in a murine model of hindlimb ischemia. Finally, histological assessment of RAGE levels in both ischemic and non-ischemic tissues was performed.Conclusions: Our RAGE-targeted probe demonstrated an average size of 450 nm, a Kd of 340-390 nM, rapid blood clearance, and a 3.4 times greater PET uptake in ischemic RAGE-expressing hindlimbs than their non-ischemic counterpart. We successfully demonstrated increased RAGE expression in a murine model of hindlimb ischemia and the feasibility for non-invasive examination of cellular, tissue, and whole-body RAGE levels with a molecularly targeted tracer.

Production and application of HMGB1 derived recombinant RAGE-antagonist peptide for anti-inflammatory therapy in acute lung injury

Acute lung injury (ALI) is an inflammatory lung disease caused by sepsis, infection, or ischemia-reperfusion. The receptor for advanced glycation end-products (RAGE) signaling pathway plays an important role in ALI. In this study, a novel RAGE-antagonist peptide (RAP) was produced as an inhibitor of the RAGE signaling pathway based on the RAGE-binding domain of high mobility group box-1 (HMGB1). Recombinant RAP was over-expressed and purified using nickel-affinity chromatography. In lipopolysaccharide- or HMGB1-activated RAW264.7 macrophage cells, RAP reduced the levels of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). RAP decreased the levels of cell surface RAGE and inhibited the nuclear translocation of nuclear factor-κB (NF-κB). These results imply that RAP decreases RAGE-mediated NF-κB activation and subsequent inflammatory reaction. For in vivo evaluation, RAP was delivered to the lungs of ALI-model animals via intratracheal administration. As a result, RAGE was down-regulated in the lung tissues by pulmonary delivery of RAP. Consequently, TNF-α, IL-6, and IL-1β were also reduced in broncoalveolar lavage fluid and the lung tissues of RAP-treated animals. Hematoxylin and eosin staining indicated that inflammation was decreased in RAP-treated animals. Collectively, these results suggest that RAP may be a useful treatment for ALI.

Effect of Linguizhugan decoction on neuroinflammation and expression disorder of the amyloid β‑related transporters RAGE and LRP‑1 in a rat model of Alzheimer's disease.

Linguizhugan decoction (LGZG), a notable prescription in Traditional Chinese Medicine, is a classical formula for the treatment of Alzheimer's disease (AD), inflammatory injury and fluid retention. The present study aimed to investigate the neuroprotective effect of LGZG on an amyloid β (Aβ)-induced AD rat model. Sprague-Dawley rats were administered with Aβ1-42 to induce AD and inflammatory responses, and subsequently with LGZG (4.8, 2.4 or 1.2 g/kg), donepezil (2 mg/kg) or distilled water for 30 consecutive days. Learning and memory behaviors were evaluated via Morris water maze test. The neuronal impairment of AD rats was observed via hematoxylin-eosin staining. The levels of pro-inflammatory cytokines, and Aβ in the brain tissue were detected with ELISA kits. Protein expression levels of mitogen-activated protein kinase and nuclear factor-κB signalling were measured by western blot analysis. The expression of lipoprotein receptor-related protein-1 (LRP-1) and receptor for advanced glycation endproducts (RAGE) in the brain were detected by western blot analysis, reverse transcription-quantitative polymerase chain reaction and immunohistochemistry analysis. LGZG was demonstrated to significantly improve learning and memory ability, and ameliorate neuroinflammation in AD rats. LGZG increased the levels of LRP-1 and decreased the levels of RAGE. Furthermore, the present results demonstrated that LGZG treatment significantly inhibited MAPK and NF-κB signalling, and reduced the production of pro-inflammatory cytokines and Aβ accumulation in AD rats. LGZG exhibited a potential protective effect on Aβ1-42-induced AD by regulating Aβ transportation, and inhibiting RAGE/MAPK and NF-κB signalling. These results suggest that LGZG may be considered for the treatment of AD.

Therapeutic Effectiveness of Anti-RAGE Antibody Administration in a Rat Model of Crush Injury

Crush injury patients often have systemic inflammatory response syndrome that leads to multiple organ failure. Receptor for advanced glycation endproducts (RAGE) functions as a pattern recognition receptor that regulates inflammation. We evaluated the effects of anti-RAGE antibody in a crush injury model. Pressure was applied to both hindlimbs of rats for 6 h by 3.0-kg blocks and then released. Animals were randomly divided into the sham (RAGE-Sh) group, crush (RAGE-Ctrl) group or anti-RAGE antibody-treated crush (RAGE-Tx) group. Samples were collected at 3, 6 and 24 h after releasing pressure. In the RAGE-Ctrl group, fluorescent immunostaining in the lung showed upregulated RAGE expression at 3 h. The serum soluble RAGE (sRAGE) level, which reflects the amount of RAGE expression in systemic tissue, increased at 6 h. Serum interleukin 6 (IL-6; systemic inflammation marker) increased immediately at 3 h. Histological analysis revealed lung injury at 6 and 24 h. Administration of anti-RAGE antibody before releasing compression inhibited upregulated RAGE expression in the lung alveoli, suppressed RAGE-associated mediators sRAGE and IL6, attenuated the lung damage and improved the 7-day survival rate. Collectively, our results indicated that the use of anti-RAGE antibody before releasing compression is associated with a favourable prognosis following crush injury.

Statins decrease vascular epithelial growth factor expression via down-regulation of receptor for advanced glycation end-products.

AimsStatins, inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase, possess pleiotropic effects that have been extended to modulation of various cellular behaviors. This study aimed to examine whether statins modulate vascular endothelial growth factor A (VEGF-A) expression in human retinal pigment epithelium (RPE) cells.Main methodsHuman RPE cells (h1RPE7), damaged by hydroquinone (HQ) + advanced glycation endproducts (AGE) in an in vitro AMD model, were treated with atorvastatin or lovastatin for 24 h. The expression of VEGF-A and receptor for AGE (RAGE) was evaluated by real-time RT-PCR. VEGF-A secretion was measured by ELISA. To investigate the impact of RAGE on VEGF-A expression, small interfering RNA (siRNA) for RAGE (siRAGE) was introduced into h1RPE7 cells and VEGF-A expression was measured by real-time RT-PCR. Deletions of VEGF-A and RAGE promoters were performed and transcriptional activities were measured after the addition of statins to HQ + AGE-damaged RPE cells.Key findingsThe mRNA levels of VEGF-A and RAGE and the levels of VEGF-A in the culture medium were increased by HQ + AGE. Both atorvastatin and lovastatin attenuated HQ + AGE-induced VEGF-A and RAGE expression. These statins also decreased VEGF-A levels in the culture medium. RNA interference of RAGE attenuated the up-regulation of VEGF-A in the HQ + AGE treated cells. The deletion analysis demonstrated that these statins attenuated RAGE promoter activation in HQ + AGE-damaged RPE cells.SignificanceStatins attenuated HQ + AGE-induced VEGF expression by decreasing RAGE expression. As VEGF is an important factor in developing wet AMD, statins could decrease the risk of wet-type AMD and be used as preventive medicines.

Association of S100 calcium-binding protein A12, receptor for advanced glycation endproducts, and nuclear factor-κB expression with inflammation in pulp tissues from tooth caries.

S100 calcium-binding protein A1 (S100A12) is a pro-inflammatory molecule which is secreted during inflammation and induces chemotaxis and the production of pro-inflammatory cytokines via interaction with receptor for advanced glycation endproducts (RAGE) and subsequent, activation of nuclear factor-κB (NF-κB). The present study was designed to determine the expression levels of S100A12, RAGE, and NF-κB in the inflamed pulp of carried teeth. In the present study, mRNA from 50 inflamed pulp and 50 healthy pulp were used for expression studies using real-time polymerase chain reaction. The expression levels of S100A12, RAGE, and NF-κB were compared between inflamed and healthy tissues. The results revealed that the expression of S100A12, but not of RAGE or NF-κB, was significantly decreased in inflamed pulp when compared to healthy pulp. mRNA levels of RAGE were also increased in the inflamed pulp taken from men when compared with women. The results suggest that S100A12 does not participate in the induction of inflammation in dental pulp. However, RAGE can participate in the inflammation in the pulp of males.

AGEs, RAGEs and s-RAGE; friend or foe for cancer.

Impaired awareness of glycation biology in cancer initiation and progression is one of the fundamental reasons for its meticulous investigation of the molecules involved in signalling pathway. Glycation of biological macromolecules results in the progression of advanced glycation end-products (AGEs) that proliferates the process of carcinogenesis by activation of transcription factors and release of cytokines. The receptor for advanced glycation end-products (RAGEs) with the binding of its different ligands like; AGEs, HMGB1 and S100 activate the signalling arrays. The activation of downstream signalling pathway ultimately leads to the pathophysiological conditions of diabetes, ageing, neurological disorders and cancers as well as a result of the activation of transcription factors which is discussed in the main body text of this review. However, there might be a likelihood of the positive effect of the HMGB1 and S100 proteins in cancer. Still, some untouched mechanisms might be responsible for the establishment of the function of AGE-RAGE or AGE-sRAGE axis activation that leads to the friend-foe association with the cancers. The levels of RAGE and s-RAGE may be a useful biomarker of ligand-RAGE pathway activation and cancer. Thus, the possibility of providing a potential complement to carcinogenesis is very high which might be an interesting target for therapeutic interventions. This article is an insightful assessment on AGE, RAGE and s-RAGE for its possible role in cancer onset and progression. The novel therapeutic targets for cancer prevention or inhibition are also explained in brief in relation to AGE and RAGE.

S100A8 inhibits PDGF-induced proliferation of airway smooth muscle cells dependent on the receptor for advanced glycation end-products

BackgroundAirway remodeling is a key feature of asthma, characterized by increased proliferation of airway smooth muscle cells (ASMCs). S100A8 is a calcium-binding protein with a potential to regulate cell proliferation. Here, the effect of exogenous S100A8 protein on the proliferation of ASMCs induced by platelet-derived growth factor (PDGF) and the underlying molecular mechanism was investigated.MethodsRat ASMCs were cultured with or without a neutralizing antibody to the receptor for advanced glycation end-products (RAGE), a potential receptor for S100A8 protein. Purified recombinant rat S100A8 protein was then added into the cultured cells, and the proliferation of ASMCs induced by PDGF was detected by colorimetric-based WST-8 assay and ampedance-based xCELLigence proliferation assay. The expression levels of RAGE in ASMCs were analyzed using western blotting assay.ResultsResults showed that exogenous S100A8 inhibited the PDGF-induced proliferation of rat ASMCs in a dose-dependent manner with the maximal effect at 1 μg/ml in vitro. Furthermore, when ASMCs was pre-treated with anti-RAGE neutralizing antibody, the inhibitory effect of S100A8 on PDGF-induced proliferation was significantly suppressed. In addition, neither the treatment with S100A8 or PDGF alone nor the pre-treatment with rS100A8 followed by PDGF stimulation affected the expression levels of RAGE.ConclusionsOur study demonstrated that S100A8 inhibits PDGF-induced ASMCs proliferation in a manner dependent on membrane receptor RAGE.

Clinical Implications of High-mobility Group Box-1 (HMGB1) and the Receptor for Advanced Glycation End-products (RAGE) in Cutaneous Malignancy: A Systematic Review.

Inflammation and the immune system play a role in the development and progression of melanoma, basal cell carcinoma (BCC), and squamous cell carcinoma (SCC). The pro-inflammatory and tumor-promoting effects of the high-mobility group box-1 (HMGB1) protein and the receptor for advanced glycation end products (RAGE) have been investigated in these cutaneous malignancies. The clinical implication of these molecules is not fully described. The National Library of Medicine database was searched for articles addressing the clinical relevance of HMGB1 and RAGE in melanoma, BCC, and SCC. This systematic review includes nine articles, with six summarizing RAGE in cutaneous malignancies and three involving HMGB1. RAGE has been found to be up-regulated in SCC lesions, as well as melanoma. Levels of RAGE were highest in stage IV melanomas. Lower levels of soluble RAGE have been associated with poor overall survival in melanoma. Sporadic extracellular expression of HMGB1 was evident in BCC and SCC lesions, which could be released by necrotic tumor cells. HMGB1 was found to be a prognostic marker in melanoma, and HMGB1 levels were elevated in patients who were non-responders to ipilimumab treatment. HMGB1 and RAGE could serve as potential prognostic markers or therapeutic targets in treating melanoma, BCC, and SCC, but further research regarding the clinical utility of the HMGB1-RAGE axis in cutaneous malignancies is warranted.

Increased Expression of the Receptor for Advanced Glycation End-Products (RAGE) Is Associated with Advanced Breast Cancer Stage

Background: The receptor for advanced glycation end-products (RAGE) is a multiligand transmembrane receptor that is overexpressed in various pathological conditions including cancers. However, the expression pattern of RAGE in breast cancer tumors is still not completely clear. Methods: In this study, we investigated the expression levels of RAGE in 25 fresh-frozen breast cancer samples and corresponding noncancerous tissue samples collected from breast cancer patients, by real-time polymerase chain reaction (PCR). Additionally, we performed immunohistochemistry on breast cancer specimens. Results: The results indicate a high expression of the RAGE-encoding gene in the cancerous tissues. RAGE expression at the mRNA and protein levels was statistically significantly up-regulated in advanced-stage and triple-negative breast tumors and node-positive tissues compared with other tissues (p < 0.001). A significant association between RAGE expression and tumor size was observed (p = 0.029). Conclusions: Overexpression of RAGE in advanced-stage tumors may be a useful biomarker for diagnosis and the prediction of breast cancer progression.

(870) - Elevated Plasma PAI-1 Levels in the Donor Are Associated with the Development of Primary Graft Dysfunction in Lung Transplant Recipients

Primary graft dysfunction (PGD) is the main cause of early morbidity and mortality following lung transplantation. Early prediction of PGD would allow earlier interventions and better patient outcomes. Clinical factors, such as cigarette smoking, have been identified in the donor that increase the risk of PGD. Biological factors may also contribute. Elevated recipient plasma levels of plasminogen activator inhibitor-1 (PAI-1) and the receptor for advanced glycation end-products (RAGE) are known to be associated with a higher risk of developing PGD. However, these and other biomarkers in the donor plasma have not yet been shown to contribute to the risk of PGD. We tested the hypothesis that elevated donor plasma levels of PAI-1 and RAGE would be associated with recipient PGD. We carried out a prospective unmatched case control study for double lung transplants between June 2014 and September 2015. The primary outcome was donor plasma levels of PAI-1 and RAGE prior to organ recovery. We compared 12 recipients who developed PGD Grade 2 or 3 by ISHLT criteria within 72 hours postoperatively, with 13 recipients who did not. PaO2/FiO2 was measured concurrent with chest radiograph upon ICU arrival, 24, 48 and 72 hours postoperatively. Plasma biomarker levels in duplicate were compared between cases and controls using the Wilcoxon rank-sum test. Donor plasma levels of PAI-1 were significantly higher in those recipients who developed grade 2 or 3 PGD compared to those who did not (p=0.03) (Table). Plasma levels of PAI-1 remained significantly higher at 24 hours in the recipients that developed PGD compared to the non-PGD group (11.57 vs 1.95 ng/mL, p<0.001). RAGE was not statistically different between groups. Donor systemic inflammatory responses as reflected by significantly elevated plasma levels of PAI-1 may contribute to the risk of developing PGD. Rapid biomarker assessment may assist in donor lung selection and early risk stratification.Tabled 1Median donor plasma levels of PAI-1 and RAGE in cases versus controlsDonor Plasma BiomarkerCases (n=12) Median (IQR)Controls (n=13) Median (IQR)P valuePAI-1 (ng/mL)2.73 (2.07-5.51)1.39 (1.07-2.89)0.03RAGE (pg/mL)1061 (475 - 1930)679 (400-747)0.11IQR, interquartile range; PAI-1, plasminogen activator inhibitor-1; RAGE, receptor for advanced glycation end-products. Open table in a new tab

Plasma Levels of Receptor for Advanced Glycation End-Products and High-Mobility Group Box 1 in Patients With Pulmonary Hypertension.

An increase of pulmonary artery pressure in patients with pulmonary hypertension (PH) results in right ventricular failure and ultimately death. High-mobility group box 1 (HMGB1), a nuclear protein, acts as a pro-inflammatory cytokine by activating receptor for advanced glycation end-products (RAGE) in the extracellular environment. The purpose of this study was to examine the clinical significance of circulating levels of HMGB1 and RAGE in patients with PH. Plasma levels of HMGB1 and soluble RAGE were measured in 27 patients with PH (14 with pulmonary arterial hypertension [PAH], 13 with chronic thromboembolic pulmonary hypertension [CTEPH]) and 30 normal subjects as control. There was no difference in the plasma levels of HMGB1 between the PH patients and the control subjects. However, plasma levels of soluble RAGE were significantly higher in the patients with PH than in the controls (P < 0.001). Plasma soluble RAGE levels were higher in PAH (P < 0.001) and CTEPH (P < 0.0001) than in the controls. In addition, there was a statistically significant positive correlation between pulmonary artery pressure and plasma levels of soluble RAGE (r = 0.403, P < 0.0001). In the CTEPH patients, soluble RAGE levels were reduced after balloon pulmonary angioplasty (P < 0.001). Plasma levels of soluble RAGE, but not HMGB1, might be a novel marker that reflects the pathological condition in patients with PH.

Overexpression of HMGB1 Receptor RAGE Is Associated with Worse Clinical Outcome in Patients with Chronic Lymphocytic Leukemia

Background: Receptor for advanced glycation end-products (RAGE) is the major receptor for inflammatory molecule HMGB1. Increased levels of RAGE are associated with poor prognosis in many types of tumours. Following engagement with extracellular HMGB1, RAGE internalises and promotes the survival and migration of cancer cells through activation of NF-kB. We have previously determined significantly increased HMGB1 levels in the plasma of CLL patients compared to healthy controls, associated with poorer clinical outcomes. HMGB1 induces differentiation of nurse like cells via RAGE and/or Toll-like receptor 9 (TLR9) harboring CLL progression and maintenance. However, it is unknown whether HMGB1/RAGE/TLR9 signaling axis can directly promote CLL B-cell survival and contribute to disease progression.We hypothesise that HMGB1 could contribute to CLL progression via direct activation of RAGE/TLR9 pathways. The aim of this study is to characterise whether activation of HMGB1-mediated pathways is associated with poorer clinical outcome in patients with CLL.Methods: HMGB1-mediated colocalisation of TLR9 and RAGE proteins was assessed in fresh primary CLL cells using the ImageStream X Mark II flow cytometer and fluorescent microscopy. Expression of RAGE and TLR9 were determined by immunohistochemistry on tissue microarrays (TMAs), containing CLL lymph node samples (n=91). The correlation between RAGE and TLR9 with other established CLL markers were analysed by both Spearman's multiple correlation and chi-square analyses. The association between protein expression and overall survival (OS) was analysed by uni- and multivariate cox regression analysis on both continuous and categorical variables to increase statistical robustness. The test/validation set-defined data cut points were determined by X-tile software.Results: HMGB1-mediated internalisation of RAGE and its colocalisation with TLR9 in primary CLL-B-cells were detected qualitatively by fluorescent microscopy and quantitatively by ImageStream X Mark II flow cytometry, demonstrating that HMGB1 directly activates both RAGE and TLR9 in CLL B-cells.RAGE and TLR9 proteins are heterogeneously expressed in CLL lymph nodes. Multiple correlation analysis identified a significant correlation between RAGE and TLR9 receptor expression (p<0.0001) suggesting they have co-linked functions. RAGE expression positively correlates with levels of Ki67 and positivity of ZAP70 (p<0.0001; p<0.05), and negatively correlates with the median OS rates, suggesting that higher expression of RAGE is associated with CLL cell proliferation and poor OS. However, RAGE expression is not correlated with CD68, a marker of tumor-associated macrophages, suggesting that increased RAGE protein may be mainly expressed by CLL B-cells.We analysed the prognostic effect of RAGE, TLR9, Ki67 and age individually by continuous univariate cox regression analysis and found that only the age of CLL patients is an independent prognostic marker for this cohort of CLL patients. Expression cut points were statistically generated using the X-tile software to define RAGE expression as high/medium/low. Increased RAGE expression (n=13) confers inferior OS (median 3 years) compared to patients with medium/low RAGE expression (n=29 and 48 respectively; median OS 7.5 years; log rank test p<0.05). There is a significant prognostic interaction between high RAGE, high Ki67 and ZAP70 categorical variables (p<0.0001), highlighting the inferior effect of overexpression of RAGE on disease outcome.Conclusions: Our data demonstrates for the first time that overexpression of HMGB1 receptor RAGE in CLL lymph nodes is associated with a worse clinical outcome in patients with CLL and positively correlated with higher expression of Ki67 and positivity of ZAP70. HMGB1 can directly activate both RAGE and TLR9 in primary CLL B-cells. We therefore propose that HMGB1 may play an important role in promoting CLL cell proliferation and survival and could be a potential target for the treatment of CLL. However, we need to further confirm our findings by combining other independent validation sets and intensive functional studies on the role of HMGB1 in CLL. DisclosuresGribben:Pharmacyclics: Honoraria; Roche/Genentech: Honoraria; Celgene: Consultancy, Honoraria; Janssen: Honoraria; Gilead: Honoraria.

Abstract 2270: RAGE-ligand signaling drives breast cancer invasion and metastasis

Abstract Background: Breast cancer is most common malignant state in women and 20% of these patients will develop metastasis during the course of their disease. Further understanding is needed of the process and mechanisms of metastasis. Our lab and others have been shown that Receptor of Advanced-Glycation End-products (RAGE) plays a role in tumorigenesis and metastasis. RAGE is highly expressed in various cancers including breast cancer and its protein levels correlate with poor patient outcome in breast cancer and other cancers. Activation of RAGE results in increased proliferation, migration and invasion of cancer cells. Further studies in mice have shown its blockade may be a therapeutic target to reduce tumor growth and the resulting metastasis. Methods: Using the breast cancer cell model (MDA-MB-231) and its organotropic sister cells lines selected in vivo for increased metastasis to lung (4175) and bone (1833), we tested the role of RAGE in driving mechanisms of metastasis in vitro and in vivo. Results: First, we demonstrated that the highly metastatic variant of 231 cells (4175 and 1833) have increased expression level of RAGE compared to MDA-MB-231 parental cells. Moreover, RAGE knockdown by shRNA in 4175 and 231 parental cells showed decreased cell invasion in transwell assays compared to control scramble shRNA. Ectopic over-expression of RAGE in parental 231 cells led to increased migratory and invasive properties compared to vector control cells, without affecting cell proliferation or viability. To explore the underlying mechanisms we probed for differences in RAGE signaling pathways including MEK/ERK, p38, AKT and SAPK/JNK; with MEK/ERK and Akt showing activation. Using chemical inhibitors of these pathways we demonstrated that the MEK/ERK, but not other pathways inhibited RAGE-driven cell invasion in transwell assays. To validate our data in vivo, we performed mammary fat pad injection of 4175 cells (RAGE and scr shRNA) in NSG mice. Tumor growth and weight was impaired in RAGE gene knockdown 4175 cells compared to scramble (scr) controls. Analysis of lung tissue retrieved from mice revealed RAGE knockdown in 4175 cells resulted in less metastasis compared to 4175 scr control cells. Conclusion: We have shown that higher RAGE levels are seen in metastatic human breast cancer BC cells. RAGE gene knockdown in metastatic BC cells reduces their invasive properties in vitro and reduces tumor progression and metastasis in vivo in NSG mice. These data highlight RAGE as a novel therapeutic target for metastatic disease in breast and other cancers. Citation Format: Taekyoung Kwak, Katherine Drews-Elger, Dekuang Zhao, Alexander Besser, Ayse Ergonul, Joyce M. Slingerland, Marc E. Lippman, Barry I. Hudson. RAGE-ligand signaling drives breast cancer invasion and metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2270. doi:10.1158/1538-7445.AM2015-2270

HMGB1 may act via RAGE to promote angiogenesis in the later phase after intracerebral hemorrhage

Following intracerebral hemorrhage (ICH), high-mobility group box 1 protein (HMGB1) may promote vascular remodeling. Whether HMGB1 supports angiogenesis after ICH is unclear, as are the receptors and downstream signaling pathway(s) involved. We used the rat model of collagenase-induced ICH to determine whether HMGB1 acts via the receptor for advanced glycation end-products (RAGE) to upregulate vascular endothelial growth factor (VEGF), a potent mitogen of endothelial cells and key regulator of normal and abnormal angiogenesis in the late phase of injury. At 3d after ICH induction, rats were treated with saline, ethyl pyruvate (EP) or N-benzyl-4-chloro-N-cyclohexylbenzamide (FPS-ZM1). ICH induced the movement of HMGB1 from the nucleus into the cytoplasm. Levels of HMGB1 and RAGE in the ipsilateral striatum increased within a few days of induction and continued to rise for 7–14d afterward. By 14d after induction, levels of VEGF and vessel density were higher than in the Sham group. Administering EP 3days after ICH induction prevented much of the stroke-induced increases in vessel density and in expression of HMGB1, RAGE, and VEGF. Administering FPS-ZM1 after ICH blocked much of the stroke-induced increases in vessel density and VEGF expression. Our results suggest that after ICH, HMGB1 may upregulate VEGF in the ipsilateral striatum predominantly via RAGE. Hence, targeting the HMGB1/RAGE signaling pathway may help reduce inappropriate angiogenesis after ICH.

Increased serum levels of LL37, HMGB1 and S100A9 during exacerbation in COPD patients

Chronic obstructive pulmonary disease (COPD) is a severe and progressive lung disease characterised by destruction of lung parenchyma and chronic airway inflammation [1]. A major cause of COPD is chronic exposure to noxious gases and particles, including cigarette smoke. During exacerbation, COPD patients experience a worsening of symptoms that coincides with increased inflammation and accelerated decline in lung function, resulting in a decrease in quality of life and increased healthcare costs. Approximately half of the COPD exacerbations causing hospitalisation are associated with respiratory viral and/or bacterial infections [2]. However, the underlying mechanisms causing COPD exacerbations are unknown [3]. Molecules derived from viruses and bacteria during airway infection can trigger the activation of pattern recognition receptors (PRRs) on lung structural and innate immune cells, and may thus contribute to the aggravation of inflammation during COPD exacerbations [1]. Interestingly, damage-associated molecular patterns (DAMPs) released from damaged or necrotic cells are also known to activate PRRs, including Toll-like receptors (TLRs) and the receptor for advanced glycation end-products (RAGE) [4]. A role for DAMPs has been proposed in the pathogenesis of COPD, as various DAMPs have been found to be increased in lung fluids and serum of COPD patients [5, 6]. Furthermore, Ager , the gene encoding RAGE, has been identified by genome-wide association studies as a susceptibility gene for COPD [7, 8]. Moreover, the serum levels of soluble RAGE (sRAGE), a decoy receptor for RAGE, were shown to be significantly lower in COPD patients, while the RAGE ligand EN-RAGE (also known as S100 calcium-binding protein (S100)A12) was significantly higher in COPD patients compared with smoking and nonsmoking controls [9]. It is currently unknown, however, whether DAMPs play a role in COPD exacerbations. Here, we hypothesised that the release of DAMPs is increased during exacerbations of COPD. Serum levels of the RAGE-activating DAMPs LL37, HMGB1 and S100A9 are increased during exacerbation in COPD patients http://ow.ly/Idh3r