Overexpression Of Receptor For Advanced Glycation End-products Research Articles

Sex-dependent deterioration of cardiac function and molecular alterations in age- and disease-associated RAGE overexpression

Elevated expression of the receptor for advanced-glycation endproducts (RAGE) in cardiac tissue is well-known in the elderly, in diabetes mellitus, and after acute cardiac infarction or ischemia/reperfusion injuries. RAGE and its binding partners affect the clinical outcome of heart failure and may play an essential role in accelerating the functional decline in cardiovascular aging. Therefore, hearts of wild-type (WT) C57black6/N and cardiac-specific RAGE-overexpressing transgenic (TR) mice were analyzed for their function by ultrasound at young (4–5 months) and old (22–23 months) ages. Transgenic mice exhibit significantly increased systolic (LVD-sy) and diastolic (LVD-di) diameters of their left ventricles. The left ventricular ejection fraction (EF) was significantly reduced in young male TR mice. Omics of the heart did not reveal direct activation of cytokine-induced inflammation. Instead, energy metabolism-associated genes were enriched in downregulated transcripts and proteins of TR animals, causing decreased ATP production. In a sex-specific manner, there was a reduced expression of the four-and-a-half LIM-domains protein 2 (FHL2). In conclusion, transgene-induced RAGE overexpression, as a model for age- and disease-associated RAGE alteration, leads to a sex-dependent EF decline, in which FHL2 and energy depletion might play crucial roles.

Increased Levels of Soluble RAGE in CLL Patient Plasma Has a Protective Effect on CLL Disease Progression

Background: Receptor for advanced glycation end-products (RAGE) is the primary receptor for the inflammatory molecule HMGB1. We previously demonstrated that HMGB1 differentiates nurse like cells (NLC), and that increased levels HMGB1 in the plasma of CLL patients and overexpression of RAGE in CLL lymph nodes is associated with a poorer clinical outcome. Moreover, we have shown that HMGB1 induced activation of NF-κB in primary CLL cells in vitro . Soluble RAGE (sRAGE) is a reported antagonistic decoy receptor of plasma HMGB1, and limits chronic inflammation. The pool of plasma sRAGE originates from 2 main sources - alternative splicing to produce endogenous secreted RAGE (esRAGE), or from surface RAGE ectodomain shedding catalyzed by ADAM10. In a healthy setting, HMGB1 and sRAGE plasma levels are inversely correlated, with increased levels of sRAGE limiting inflammatory responses. RAGE shedding in response to saturating levels of ligand (i.e. HMGB1) establishes a negative feedback loop, dampening inflammatory signaling by sequestering RAGE ligands. In chronic disease, the HMGB1-sRAGE inverse relationship is frequently reversed. The functional impact of sRAGE is poorly documented in cancer, and its role in CLL is currently unknown.We hypothesize that sRAGE could have a protective effect in CLL and could dampen HMGB1-mediated disease progression. The aim of this study is to characterise the clinical significance of sRAGE in CLL and investigate whether RAGE undergoes membrane shedding to confer a protective effect in CLL.Methods: sRAGE plasma levels were measured by ELISA in 54 CLL patients and 15 healthy controls. sRAGE protein mass was assessed by Western blotting and compared to matched patient ADAM10 whole cell expression. ADAM10 inhibitor studies and HMGB1-induced RAGE shedding was performed in RAGE-transfected HEK293 cell line and primary CLL cells; sRAGE production was analyzed by ELISA and surface RAGE expression determined by flow cytometry. sRAGE containing media was produced by HEK293 cell line transfected with esRAGE isoform gene sequence; sRAGE containing media (2ng/ml) was used to assess the effect on primary CLL NLC differentiation.Results: sRAGE plasma levels were significantly higher in CLL patients compared to healthy controls (median 60.0pg/ml ± 79.5 interquartile range and 31.0pg/ml ±46.9, respectively; P <0.01), but fell below levels capable of full HMGB1 inhibition for the same patient cohort (72.2ng/ml ±111.4). Absence of a negative correlation between HMGB1 and sRAGE suggested a breakdown in the negative feedback loop in CLL, compared to the classical inverse trend observed for the healthy controls. Patients with high levels of plasma sRAGE had an increased time to first treatment (P=0.0134; n=26) and sRAGE levels were increased in patients who had reached remission following treatment compared to pre-treatment samples, suggesting that sRAGE provides a protective effect on CLL disease progression.CLL plasma sRAGE is of shorter length than cellular full-length RAGE and esRAGE from primary CLL cells, suggesting sRAGE originates from ectodomain shedding. ADAM10 expression, the main RAGE sheddase, is independent of sRAGE plasma levels assessed by Western blotting. ADAM10 stimulation with ionomycin induced RAGE ectodomain shedding into cell culture medium in RAGE-transfected HEK293 cell line (2270.0ng/ml) compared to untreated cells, without altering surface RAGE or ADAM10 expression. Ionomycin-induced RAGE shedding was abrogated by pre-treatment with ADAM10 inhibitor, GI254023X (45.5ng/ml). Ionomycin induced apoptosis in primary CLL cells, so could not be investigated for RAGE shedding, but our data demonstrates that saturating HMGB1 levels induces RAGE shedding in CLL. Finally, sRAGE-containing media did not reduce primary CLL NLC differentiation in vitro after 1-2 weeks culture.Conclusions: Our data demonstrates for the first time that plasma sRAGE limits CLL disease progression by prolonging time to first treatment and sustaining stable disease following treatment. sRAGE originates from ectodomain shedding, is dependent on ADAM10 and inducible by HMGB1. Although sRAGE failed to block NLC differentiation in vitro, it contributes to the extracellular mileu ‘cocktail’ shaping the CLL microenvironment. We therefore propose that HMGB1 and sRAGE levels could be useful plasma markers for inflammatory microenvironment and therapy monitoring. DisclosuresGribben:Acerta: Honoraria; Pharmacyclics: Honoraria; Celgene: Honoraria; TG Therapeutics: Honoraria; Karyopharm: Honoraria; Janssen: Honoraria; Kite: Honoraria; Abbvie: Honoraria; Genentech/Roche: Honoraria.

Downregulation of RAGE Inhibits Cell Proliferation and Induces Apoptosis via Regulation of PI3K/AKT Pathway in Cervical Squamous Cell Carcinoma.

AimThe receptor for advanced glycation endproducts (RAGE) expression has been reported to be implicated with cancer development. In this study, the role of RAGE in the regulation of cervical squamous cancer cell proliferation, apoptosis and the mechanism of RAGE involved in the biological behaviors were explored.MethodsThe RAGE expression was overexpressed or downregulated by lentivirus transfection. The effect of RAGE expression on cell proliferation was explored by CCK-8, MTT, and BrdU assay, and the effect of RAGE on tumor development was confirmed by the xenograft mouse model along with the immunohistochemistry stain of proliferating cell nuclear antigen (PCNA). Apoptosis was investigated by flow cytometry and TUNEL assay. Western blotting was performed to investigate the expression of possible proteins, including Bax, Bcl-2, PI3K, p-PI3K, AKT, and p-AKT.ResultsOverexpression of RAGE promoted proliferation of cervical squamous cancer cell and increased PCNA expression. In the meantime, RAGE overexpression inhibited cell apoptosis along with a decrease of Bax/Bcl-2 ratio, and induction of PI3K/AKT activation. The in vivo results showed that overexpression of RAGE enhanced tumor growth. Conversely, knockdown of RAGE exhibited opposed effects on cervical cancer cells and xenograft mouse model. Furthermore, RAGE inhibitor FPS-ZM1 effectively inhibited SiHa cell viability and PCNA expression, and increased cell apoptosis and Bax/Bcl-2 ratio. Moreover, PI3K inhibitor LY294002 effectively inhibited activation of PI3K and AKT, and further repressed RAGE overexpression-induced cell proliferation and apoptosis inhibition.ConclusionRAGE promotes the growth ability of cervical squamous cell carcinoma by inducing PCNA expression and inhibiting cell apoptosis via inactivation of the PI3K/AKT pathway.

Plasma amyloid beta level changes in aged mice with cognitive dysfunction following sevoflurane exposure.

Previous studies have stated that cognitive impairment induced by anesthetics was associated with amyloid beta (Aβ). However, few researchers have investigated the transport of Aβ inside and outside of the brain. We attempted to probe the effects of sevoflurane on cognitive functions, the plasma Aβ, and transporters of Aβ in aged mice. The receptor for advanced glycation end-products (RAGE) is an Aβ influx protein, and Low-density lipoprotein receptor-related protein-1 (LRP-1) is an Aβ efflux protein. Aged mice were divided into the control group and the sevoflurane group. The mice were exposed to 100% oxygen or 2.5% sevoflurane for 2 h. The abilities of spatial learning and memory in mice were tested using the Morris water maze. Aβ concentrations of plasma were measured with enzyme-linked immunosorbent assay kits. The RAGE and LRP-1 gene levels in the brain were assessed with quantitative polymerase chain reaction, and the protein levels were determined by western blot analysis. The locations of RAGE in the brain were confirmed via immunofluorescence. In the sevoflurane group mice, the escape latency was increased on the 5th day of training, and the time spent in the target quadrant was decreased on the 7th day after anesthesia. Sevoflurane reduced the concentration of plasma Aβ1-40. In addition, sevoflurane increased both gene and protein levels of RAGE in the brain, and increased RAGE proteins co-localized with the hippocampal vascular endothelial cells. RAGE over-expression in the hippocampal vascular endothelial cells possibly resulted in the excessive transport of the plasma Aβ1-40 into the brain after treatment with sevoflurane, which was associated with sevoflurane-induced cognitive dysfunction in aged mice.

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.

AGE-RAGE interaction in the TGFβ2-mediated epithelial to mesenchymal transition of human lens epithelial cells.

Basement membrane (BM) proteins accumulate chemical modifications with age. One such modification is glycation, which results in the formation of advanced glycation endproducts (AGEs). In a previous study, we reported that AGEs in the human lens capsule (BM) promote the TGFβ2-mediated epithelial-to-mesenchymal transition (EMT) of lens epithelial cells, which we proposed as a mechanism for posterior capsule opacification (PCO) or secondary cataract formation. In this study, we investigated the role of a receptor for AGEs (RAGE) in the TGFβ2-mediated EMT in a human lens epithelial cell line (FHL124). RAGE was present in FHL124 cells, and its levels were unaltered in cells cultured on either native or AGE-modified BM or upon treatment with TGFβ2. RAGE overexpression significantly enhanced the TGFβ2-mediated EMT responses in cells cultured on AGE-modified BM compared with the unmodified matrix. In contrast, treatment of cells with a RAGE antibody or EN-RAGE (an endogenous ligand for RAGE) resulted in a significant reduction in the TGFβ2-mediated EMT response. This was accompanied by a reduction in TGFβ2-mediated Smad signaling and ROS generation. These results imply that the interaction of matrix AGEs with RAGE plays a role in the TGFβ2-mediated EMT of lens epithelial cells and suggest that the blockade of RAGE could be a strategy to prevent PCO and other age-associated fibrosis.

Receptor for advanced glycation end-products (RAGE) is overexpressed in human osteosarcoma and promotes the proliferation of osteosarcoma U-2OS cells in vitro.

Osteosarcoma (OS) is an aggressive cancer of the long bones, and usually affects children and young adults. The receptor for advanced glycation end-products (RAGE) has recently been recognized as an oncogenic receptor that binds to different ligands, and promotes the progression of various cancers. However, little is known about the association between RAGE and the pathogenesis of OS. In this study, we first examined the expression of RAGE in OS tissues using immunohistochemical staining, western blotting, and reverse transcription quantitative polymerase chain reaction. We then determined the influence of the overexpressed RAGE on the proliferation of U-2OS cells in vitro. The results showed that RAGE was overexpressed in OS tissues compared with peritumor tissues, at both the mRNA and protein levels, and there was a significant association between overexpressed RAGE and clinicopathological characteristics, such as clinical stage and distant metastasis. Moreover, the overexpression of RAGE in U-2OS cells significantly promoted their proliferation in vitro. In conclusion, this study indicated that RAGE is overexpressed in OS tissue and promotes the proliferation of U-2OS cells. These data imply that RAGE promotes the growth of OS, and is a potential diagnostic biomarker and therapeutic target for the disorder.

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 805: Subcellular distribution of RAGE affects its functions in melanoma growth and progression

Abstract The receptor for advanced glycation end-products (RAGE) is known to act as a central driver of tumorigenesis by sustaining a chronic inflammatory tumor microenvironment. Until to date, RAGE has been exclusively described as a cell surface receptor being activated upon engagement with its various extra-cellular ligands, e.g. S100B, S100A8/A9, HMGB1, and others. This study aimed at elucidating the functional role of RAGE and its isoforms depending on their subcellular distribution in the context of melanoma development, growth and progression. Therefore, various in vitro models using melanoma cells or melanocytes and melanoma mouse models as well as tissue-microarrays representing human specimens of malignant melanoma and benign nevi were applied. The expression analyses revealed an overexpression of RAGE in melanoma cells compared to melanocytes/nevocytes. Moreover, RAGE protein was found to be localized primarily in the nucleus of melanocytes/nevocytes whereas a predominant cell surface/cytoplasmic localization of RAGE is observed in melanoma cells. Nuclear translocation of RAGE depends on the nuclear transport machinery and site-directed mutagenesis of predicted DNA binding sites within the RAGE protein was applied in order to study its functional role in the nucleus. Furthermore, knockdown of RAGE indicated its central function in apoptosis induction. In conclusion, RAGE and its isoforms are overexpressed and aberrantly localized in malignant melanoma cells compared to melanocytes/nevocytes. Our data point towards a novel tumor-protective function of RAGE in melanoma development depending on its nuclear localization. Citation Format: Maike Reith, Wolf-Henning Gebhardt, Kathrin Tarnanidis, Nikolaus B. Wagner, Kristian Ikenberg, Coretta Kehrel, Jochen Utikal, Christoffer Gebhardt. Subcellular distribution of RAGE affects its functions in melanoma growth and progression. [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 805. doi:10.1158/1538-7445.AM2015-805

Overexpression of the Receptor for Advanced Glycation Endproducts (RAGE) is associated with poor prognosis in gastric cancer.

BackgroundThe receptor for advanced glycation endproducts (RAGE) is an oncogenic multidisciplinary trans-membranous receptor, which is overexpressed in multiple human cancers. Recently, it has been shown that RAGE is also involved in carcinogenesis and tumor invasion. In this study, we investigated the expression levels and prognostic value of RAGE in primary gastric cancers (GC).MethodsWe investigated RAGE expression in primary GC and paired normal gastric tissue by real-time quantitative RT-PCR (n = 30) and Western blotting analysis (n = 30). Additionally, we performed immunohistochemistry on 180 paraffin-embedded GC specimens, 69 matched normal specimens.ResultsRAGE was overexpressed in GC compared with the adjacent noncancerous tissues (P＜0.001), and higher RAGE expression significantly correlated with the histological grade (P = 0.002), nodal status(P = 0.025), metastasis status(P = 0.002), and American Joint Committee on Cancer stage (P = 0.020). Furthermore, upregulation of RAGE expression is an independent prognostic factor in multivariate analysis using the Cox regression model (P = 0.001).ConclusionsRAGE Overexpression may be a useful marker to predict GC progression and poor prognosis.

Metabolic syndrome and colorectal cancer: is hyperinsulinemia/insulin receptor-mediated angiogenesis a critical process?

Components of metabolic syndrome (MS) have been individually linked to colorectal cancer risk and prognosis; however, an understanding of the dominant mechanisms is lacking. Twenty-one patients (10 MS; 11 non-MS) with resectable colorectal cancer were prospectively enrolled. Patients were classified for MS by the World Health Organization criteria and tested for circulating vascular endothelial growth factor (VEGF), interleukin-6 (IL-6), insulin-like growth factor-1 (IGF-1), fasting insulin, and tumor expression of IGF-1 receptor (IGF-1R), insulin-receptor (IR) and receptor for advanced glycation end-products (RAGE). Circulating markers were re-tested 6 months after surgery. The MS group had significantly higher baseline and post-operative fasting insulin levels (p < 0.001 and 0.003). No differences were observed in circulating IL-6, VEGF, IGF-1 and free IGF-1. By immunohistochemistry (IHC), IGF-1R expression was significantly higher in tumor vs. normal tissues (p < 0.001) while IR expression showed no difference. Interestingly, 64% of tumors demonstrated high IR positivity in the vessels within or surrounding the tumor stroma, but not in the vessels away from the tumor. By reverse transcription polymerase chain reaction (RT-PCR), tumor IGF-1R over-expression (80%) was confirmed, but there was no difference between MS and non-MS patients. Tumor RAGE over-expression was found in 67% of patients and was equally distributed between the two groups. Hyperinsulinemia was the only significant factor distinguishing patients with colorectal cancer who have MS. The preferential over-expression of IR in the peri-tumoral microvessels suggests that hyperinsulinemia might contribute to colorectal cancer growth by enhancing angiogenesis.

Losartan protects liver against ischaemia/reperfusion injury through PPAR‐γ activation and receptor for advanced glycation end‐products down‐regulation

PPAR-γ has been reported to be a protective regulator in ischaemia/reperfusion (I/R) injury. The receptor for advanced glycation end-products (RAGE) plays a major role in the innate immune response, and its expression is associated with PPAR-γ activation. Several angiotensin receptor blockers possess partial agonist activities towards PPAR-γ. Therefore, this study investigated the action of losartan, particularly with regard to PPAR-γ activation and RAGE signalling pathways during hepatic I/R. Mice were subjected to 60 min of ischaemia followed by 6 h of reperfusion. Losartan (0.1, 1, 3 and 10 mg · kg⁻¹) was administered 1 h prior to ischaemia and immediately before reperfusion. GW9662, a PPAR-γ antagonist, was administered 30 min prior to first pretreatment with losartan. Losartan enhanced the DNA-binding activity of PPAR-γ in I/R. Losartan attenuated the increased serum alanine aminotransferase activity, TNF-α and IL-6 levels, and nuclear concentrations of NF-κB in I/R. GW9662 reversed these beneficial effects. Losartan caused a decrease in apoptosis as assessed by TUNEL assay, in release of cytochrome c and in cleavage of caspase-3, and these effects were abolished by GW9662 administration. Losartan attenuated not only I/R-induced RAGE overexpression, but also its downstream early growth response protein-1-dependent macrophage inflammatory protein 2 level; phosphorylation of p38, ERK and JNK; and subsequent c-Jun phosphorylation. GW9662 reversed these effects of losartan administration. Our findings suggest that losartan ameliorates I/R-induced liver damage through PPAR-γ activation and down-regulation of the RAGE signalling pathway.

Metabolic syndrome and colon cancer: Is hyperinsulinemia/insulin receptor-mediated angiogenesis a critical process?

e14004 Background: Components of the metabolic syndrome (MS) have been individually linked to colon cancer (CC) risk and prognosis. An understanding of the dominant mechanisms is lacking. Methods: 21 patients (10 MS; 11 non-MS) with resectable CC were prospectively enrolled. Patients were classified for MS by the WHO criteria and tested for circulating vascular endothelial growth factor (VEGF), IL-6, insulin-like growth factor-1 (IGF-1), fasting insulin, and tumor expression of IGF-1 receptor (IGF-1R), insulin-receptor (IR) and receptor for advanced glycation end-products (RAGE). Circulating markers were re-tested 6 months after surgery (Sx). Results: The MS group had significantly higher baseline and post-operative fasting insulin levels (p<0.001 and 0.003). No differences were observed in circulating IL-6, VEGF, IGF-1 and free IGF-1. By immunohistochemistry (IHC), IGF-1R expression was significantly higher in tumor vs. normal tissues (p<0.001) while IR expression showed no difference. Interestingly, 7 out of 11 tumors demonstrated high IR positivity in the neovessels within or surrounding the tumor stroma, but not in the vessels away from the tumor. By RT-PCR, tumor IGF-1R over-expression (80%, 12/15) was confirmed, but there was no difference between MS and non-MS patients. Tumor RAGE over-expression was found in 67% (10/15) of patients who equally distributed between the two groups (Table). Conclusions: Hyperinsulinemia was the only significant factor distinguishing CC patients with MS. The preferential overexpression of IR in the peri-tumoral microvessels suggests hyperinsulinemia may contribute to CC growth by enhancing angiogenesis. Comparisons of circulating and tissue markers between MS and non-MS patients. Serum Tissue IGF-1 (ng/L) Free IGF-1 (pg/L) Insulin (uIU/mL) Insulin-R IHC (Allred score) IGF-1R IHC (Allred score) IGF-1R PCR N pre-Sx post-Sx pre-Sx post-Sx pre-Sx post-Sx N Tumor (11) Normal (19) Peri-tumorvessel (7) Tumor (11) Normal (19) N Over-express Under-express MS MedianRange 10 144 145 458 354 12 11 16 3 4 8 7 4 7 7 0 264 111 982 678 20 34 6 7 2 2 2 Non-MS MedianRange 11 118 127 592 361 4 6 14 0 5 8 6.5 4 8 5 3 206 154 431 1037 8 15 4 7 2 2 3

Molecular Pathways Involved in RAGE Mediated Attenuation of Arteriogenesis

The receptor for advanced glycation endproducts (RAGE) has been implicated in promoting vascular dysfunction through the activation of proinflammatory cascades and the induction of oxidative stress. Chronic activation of RAGE is associated with diabetes‐related vascular complications including coronary and peripheral vascular atherosclerosis and diabetic retinopathy. We have demonstrated that RAGE activation in monocytes and macrophages also regulates vascular remodeling in response to acute ischemia in a mouse model of hind‐limb ischemia. Lentiviral‐directed overexpression of RAGE in monocytes prevents the development of collateral vessels (arteriogenesis) in response to hind‐limb ischemia. To better understand the molecular mechanisms responsible for these observations, we have undertaken a cell culture approach to address the effect of monocyte ‐specific RAGE activation on the production of proarteriogenic and proangiogenic factors under hypoxic conditions and determine how chronic RAGE activation influences receptor recycling. We have generated soluble RAGE using a baculovirus system, which will provide a valuable reagent to evaluate RAGE signaling in response to hypoxic conditions. In addition, we are generating N‐terminal GFP‐tagged versions of the RAGE receptor that will allow us to evaluate RAGE receptor recycling following activation in a macrophage cell line.

The receptor for advanced glycation end products (RAGE) sustains autophagy and limits apoptosis, promoting pancreatic tumor cell survival.

Activation of the induced receptor for advanced glycation endproducts (RAGE) leads to initiation of NF-κB and MAP kinase signaling pathways resulting in propagation and perpetuation of inflammation. RAGE knock out animals are less susceptible to acute inflammation and carcinogen induced tumor development. We have reported that most forms of tumor cell death result in release of the RAGE ligand, HMGB1. We now report a novel role for RAGE in the tumor cell response to stress. Targeted knockdown of RAGE in the tumor cell, leads to increased apoptosis, diminished autophagy and decreased tumor cell survival . In contrast, overexpression of RAGE is associated with enhanced autophagy, diminished apoptosis and greater tumor cell viability. RAGE limits apoptosis through a p53 dependent mitochondrial pathway. Moreover, RAGE-sustained autophagy is associated with decreased phosphorylation of mTOR and increased Beclin-1/VPS34 autophagosome formation. These findings demonstrate that the inflammatory receptor RAGE plays a heretofore unrecognized role in the tumor cell response to stress. Furthermore, these studies establish a direct link between inflammatory mediators in the tumor microenvironment and resistance to programmed cell death. Our data suggest that targeted inhibition of RAGE or its ligands may serve as novel targets to enhance current cancer therapies.

Short-chain aldehyde-derived ligands for RAGE and their actions on endothelial cells

The formation and accumulation of advanced glycation endproducts (AGE) have been implicated in the development of diabetic vascular complications. Their biological responses are known to be mediated by the receptor for AGE (RAGE). Recently, AGE have been proposed to be derived not only from the classical Maillard reaction but also from other pathways of sugar autoxidation and metabolism. Here, we report the identification of glyceraldehydes (Gcer)- and glycolaldehyde (Gcol)-derived AGE as RAGE ligands and their presence in vivo. The apparent dissociation constants assessed by surface-plasmon resonance (SPR) analysis with purified human RAGE proteins were 360 nM for Gcer-AGE and 1.35 μM for Gcol-AGE. The radiolabeled-ligand binding assay with RAGE-expressing COS-7 cells revealed similar association kinetics. Competitive SPR assay with antibodies specific to the respective AGE fractions demonstrated abundant existence of both Gcer- and Gcol-AGE in RAGE affinity-purified proteins from human sera. The serum contents of Gcer- and Gcol-AGE in a diabetic patient were about twice as high as those in a healthy control. Functionally, Gcer- and Gcol-AGE upregulated the endothelial cell levels of mRNA for vascular endothelial growth factor (VEGF) and the secretion of its protein product into the culture media and DNA synthesis in a dose-dependent manner. Further, these endothelial responses were augmented by RAGE overexpression. The results suggest that RAGE engagement of Gcer- and Gcol-AGE may elicit angiogenesis through the induction of autocrine VEGF, thereby contributing to the development and progression of diabetic angiopathies.

Enhanced scavenging of lipid substances is a possible effect of corticosteroids in the treatment of cholesterol crystal embolism

Cholesterol crystal embolism (CCE) is a systemic refractory disease especially prevalent amongst elderly patients suffering from atherosclerosis. Treatment of this condition remains controversial due to difficulties in diagnosis. Corticosteroid therapy may be an important treatment option despite its elusive mechanisms. To clarify the role of corticosteroid in CCE therapy, we collected the samples from six autopsied subjects with CCE, three of whom were clinically given various doses of corticosteroid to investigate stable atherosclerosis-related substances, advanced glycation end-products (AGE), and several AGE receptors such as scavenger receptor class B type 1 (SR-B1), receptor for AGE (RAGE), and galectin-3 in the liver tissues and atherosclerotic areas by immunostaining using a tissue macro-array technique. An intense expression of AGE and its receptors was identified in the enlarged Kupffer cells of CCE cases, which were given relatively high doses of corticosteroid. In addition, numerous mononuclear cells in the intimal atheromatous plaque presented strong expressions of AGE and SR-B1. In conclusion, we speculated that corticosteroid treatment for CCE may upregulate the activations, including phagocytic capacity of Kupffer cells mediated by overexpression of RAGE and scavenger receptors, resulting in efficient clearance of the lipid substances from the blood circulation released from atherosclerotic areas.

Proliferative Stimulus of Lung Fibroblasts on Lung Cancer Cells Is Impaired by the Receptor for Advanced Glycation End-Products

The receptor for advanced glycation end-products (RAGE) is highly expressed in lung tissue, especially at the site of the alveolar epithelium, but its expression is reduced in lung carcinomas. Because epithelial-mesenchymal interactions are suggested to contribute to cancer progression, we investigated the RAGE-dependent impact of fibroblasts on tumor cell growth. Cocultivation of human lung cancer cells (H358) with lung fibroblasts (WI-38) improved their proliferation in monolayer and spheroid culture models, the number of H358 cells in the S/G2 cell cycle phase increased, and there was less spontaneous cell death. Overexpression of full-length human RAGE reduced the proliferative stimulus of fibroblasts as seen in monolayers (cell number, cell cycle), spheroid cultures (spheroid size), and in a colony-forming assay compared with mock-transfected cells. Comparable results were observed by culturing H358 cells with and without RAGE overexpression in the presence of conditioned medium taken from WI-38 cells, or in response to selected growth factors, such as basic fibroblast growth factor. Moreover, we clearly showed that the fibroblast-induced proliferation correlates with activation of the p42/44 mitogen-activated protein kinase, but not with Akt kinase activation. On the basis of lung cancer as an age-related disease, we additionally proved the impact of senescent WI-38 fibroblasts. Here, we show that senescent fibroblasts improve H358 cell proliferation to the same extent as do presenescent fibroblasts. From our data, we conclude that re-expression of RAGE in lung cancer cells impairs the proliferative stimulus mediated by fibroblasts. Therefore, lung cancer progression may be enhanced by the RAGE downregulation in human lung carcinomas.

Roles of the Receptor for Advanced Glycation Endproducts in Diabetes-Induced Vascular Injury

Diabetic patients have shorter life span and poorer Quality of Life mainly due to diabetic vascular complications. Recent in vitro and in vivo studies have shown that advanced glycation endproducts (AGE) account for diabetic vascular complications through their engagement of the receptor for AGE (RAGE). In this review, we summarize our recent studies on the roles of the AGE-RAGE system in diabetes-induced vascular injury. In vitro experiments showed that AGE engagement of RAGE leads to changes in endothelial cells (EC) and pericytes, which are characteristic of diabetic microangiopathy. Diabetic RAGE transgenic mice that overexpress RAGE in vascular cells exhibited the exacerbation of the indices of nephropathy and retinopathy, and this was prevented by the inhibition of AGE formation. RAGE overexpression also caused calcium handling impairment in cardiac myocytes. In contrast to the RAGE-overexpressing mice, diabetic RAGE knockout mice showed marked improvement of nephropathy. We found that human vascular cells express a novel splice variant coding for a soluble RAGE protein and named it endogenous secretory RAGE (esRAGE). The esRAGE neutralizes AGE actions on EC and is present in human sera. Individual variations in circulating esRAGE could be a determinant for individual differences in susceptibility or resistance to the development of diabetic vascular complications. The AGE-RAGE system should be, therefore, a candidate molecular target for overcoming diabetic vascular complications.

Down-regulation of the receptor for advanced glycation end-products (RAGE) supports non-small cell lung carcinoma

The receptor for advanced glycation end-products (RAGE) is a transmembrane receptor of the immunoglobulin superfamily. Several ligands binding to RAGE have been identified, including amphoterin. Experimental studies have given rise to the discussion that RAGE and its interaction with amphoterin contribute to tumour growth and metastasis. However, none of the studies considered a differential transcription profile in cancer that might change the interpretation of the study results when comparing RAGE in tumours with histologically normal tissues. Here we show that RAGE is strongly reduced at the mRNA and even more so at the protein level in non-small cell lung carcinomas compared with normal lung tissues. Down-regulation of RAGE correlates with higher tumour (TNM) stages but does not depend on the histological subtypes, squamous cell lung carcinoma and adenocarcinoma. Subsequent overexpression of full-length human RAGE in lung cancer cells (NCI-H358) showed diminished tumour growth under some conditions. While proliferation of RAGE-expressing cells was less than that of cells expressing the cytoplasmic domain deletion mutant DeltacytoRAGE or mock-transfected NCI-H358 in monolayer cultures, RAGE cells also formed smaller tumours in spheroid cultures and in vivo in athymic mice compared with DeltacytoRAGE cells. Moreover, we observed a more epithelial growth of RAGE-expressing, but also of DeltacytoRAGE-expressing, cells on collagen layers, whereas mock NCI-H358 cells kept their tumour morphology. This observation was supported by immunofluorescence analyses demonstrating that RAGE preferentially localizes at intercellular contact sites, independent of expression of the cytoplasmic domain. Thus, down-regulation of RAGE may be considered as a critical step in tissue reorganization and the formation of lung tumours.