Dissertations / Theses on the topic 'Receptor for advanced glycation endproduct'

INTRODUCTION AGEs and ALEs (Advanced Glycoxidation/Lipoxidation End products) are covalently modified proteins that can act as pathogenic factors in several chronic diseases, like diabetes and cardiovascular diseases. These covalent adducts are formed by different mechanisms. AGEs are proteins covalently modified by reducing sugars or their oxidative degradation products, involving the Maillard reaction. ALEs are proteins modified by reactive carbonyl species (RCS) generated by lipid peroxidation. AGEs/ALEs can be the basis of many different pathologies, underlining the importance for good analytical methods for identification and characterization for the use of biomarkers, but also as a drug target. However, the identification, characterization and quantification of AGEs/ALEs remains to be very challenging due to heterogeneous precursors (sugars, lipids) leading to heterogeneous AGEs/ALEs, present in low concentrations and being very complex analytes. Various techniques to identify and characterize AGEs/ALEs have been described, making use of an isolation/enrichment step based on reactive groups, like carbonyls. However, not all AGEs/ALEs retain reactive groups and therefore can not be isolated and identified using these techniques, indicating the need for a new strategy. The strategy that has been employed in our laboratory is to use the soluble domain of the RAGE receptor, VC1, to affinity enrich AGEs. Using this approach, AGEs/ALEs will be enriched independently of the protein and type of modification. Moreover, a ligand of RAGE can be identified, which could be a potential biomarker of a disease caused by oxidative stress. RAGE is a type I cell surface receptor that is expressed in several cells, such as endothelial cells, smooth muscle cells, but also dendritic cells and T-lymphocytes and is predominantly located in the lungs. The receptor has been implicated in many different pathologies with a marked oxidative base, such as diabetes, atherosclerosis and neurodegenerative diseases. One of the pathways that can be activated is the Nf-κB pathway. The Nf-κB pathway is the ideal signaling pathway to investigate the binding and activation of RAGE by AGEs or ALEs. For this purpose, a cell line was obtained with and without overexpression of RAGE. Furthermore, the cell lines were transfected with a Nf-κB reporter gene, providing us with a fast and high-throughput assay for the evaluation of a pro-inflammatory response upon stimulation with AGEs/ALEs. AIM OF THE PROJECT The identification and characterization of AGEs/ALEs has proven to be crucial in the onset and development of many pathologies. Therefore, good analytical strategies need to be developed/optimized for better understanding of the exact nature of modification, to understand the role they play in disease progression. Identified AGEs/ALEs can serve as biomarker, as well as drug targets. The VC1 technique was proven to be a promising technique to accommodate the need for enrichment of AGEs for better characterization. The first aim of the project was therefore to investigate whether also ALEs are binder of RAGE, since they share the same structural properties than AGEs, and also have been shown to activate the Nf-κB pathway, implicating a role for receptors, like RAGE. Furthermore, to gain a deeper insight into the molecular mechanisms involved in the protein-protein engagement. Since a successful enrichment strategy was developed, the second aim of this project was focused on identifying AGEs/ALEs in biological samples. The first part was focused on oxidizing healthy human plasma in-vitro using AAPH as a radical initiator, and the incubation of plasma directly with RCS, anticipating the production of AGEs/ALEs. The VC1 technique was then used to identify which AGEs/ALEs are produced. Simultaneously, other variables during the sample preparation and analysis were optimized. As explained before, AGEs/ALEs are present in very low concentrations in biological samples, hence the need for very sensitive methods and instrumentation allowing identification. Since human serum albumin (HSA) is the main protein present in plasma, around 50-60%, and has multiple nucleophilic targets, it represents the best model for characterizing AGEs/ALEs. For this reason, the focus was on extracting HSA from plasma, using the newest generation of tribrid MS for the analysis of AGEs/ALEs in plasma samples. AGEs are ligands for RAGE, meaning, they can bind and activate the receptor, inducing a signaling pathway and pro-inflammatory response. ALEs have also been shown to induce a pro-inflammatory response; however, no specific receptor has been linked to this cellular event. Using a cell line with and without RAGE, we aimed to determine whether ALEs can bind and activate the Nf-κB pathway through RAGE. RESULTS AND DISCUSSION ALEs as binder of RAGE In order to investigate the interaction between RAGE and ALEs, different ALEs were produced in-vitro by incubating HSA with different concentrations of well-known lipid derived RCS and in particular: ACR, MDA and HNE. After 24, 48 and 72 h, aliquots of the incubation mixtures were withdrawn, and the reaction was stopped by removing the excess of RCS by ultrafiltration. Intact protein analysis by direct infusion MS was used to evaluate the extent of HSA modifications and demonstrated that by using a wide range of molar ratios and different time-points a quite wide array of ALEs for each tested RCS was generated. In order to characterize ALEs selectively enriched by RAGE, a VC1 pull-down assay was performed as previously described. HSA and HSA treated with MDA, ACR or HNE were assayed for binding to VC1-resins and to control resin. As expected, unmodified HSA was not retained by the VC1-resin. At increasing molar ratios and incubation time, higher amounts of albumin modified with MDA or ACR were eluted from the VC1 resin, with a predominance of the high molecular weight (HMW) species. The modified albumins were retained by the VC1-resin, but not by the control resin. ALEs in the reaction mixtures and those enriched by VC1 were analyzed by bottom-up MS in order to identify the PTMs and to localize the amino acid residues involved in the protein adduct formation. With regard to MDA, only di-hydropyridine adducts on lysines (DHPK), and N-2-pyrimidyl-ornithine adducts on arginines (NPO) were retained by VC1-domain. The n-propenal modifications of lysine (NPK), largely identified before enrichment, were not identified after the enrichment. ACR induced a set of modifications which were identified only after VC1 enrichment and in particular the N-(3-formyl-3,4-dehydro-piperidinyl) lysine (FDPK) modifications, the Michael adduct on cysteines, the double Michael adduct of lysines, the Michael adduct of histidine, the N-2-(4 hydroxy-tetrahydro-pyrimidyl) ornitine (propane-arginine, HTPO) and the Nε-(3-methylpyridinium)-lysine (MP-lysine). Most of the ALEs generated by HNE were found both before or after enrichment, with the exception of a few Michael adducts which were selectively retained by VC1 (not detected before enrichment). With a view to rationalizing the key factors influencing the RAGE binding of the monitored adducts, in silico studies were performed. They were focused on the adducts on arginine and lysine residues as formed by ACR and MDA since they are numerous, with a very broad range of affinity, thus allowing the development of clear structure-affinity relationships. RAGE-ligand interacting regions are characterized by a rich set of positively charged residues which can bind acidic regions of a protein. The mechanism identified using in silico studies, involves a basic amino acid at the center of carboxylic acids like glutamate and aspartate, which forms a set of ionic bridges. Once the basic amino acid is modified by ACR or MDA to an adduct with a neutral charge, the carboxylic acids become available to freely contact the RAGE positive residues. Identification of AGEs/ALEs in biological samples The VC1 technique has proven to be successful in enriching AGEs and ALEs, so the next step was to exploit this technique in biological samples. In order to identify proteins prone to be modified due to oxidative pathways, and possibly serve as biomarker, healthy human plasma was oxidized using the radical initiator AAPH. Different concentrations of AAPH and different timepoints were tested for the presence of protein carbonyl groups, an indicator for protein oxidation and possibly the formation of AGEs/ALEs. A time and concentration dependent formation of carbonyl groups is observed in plasma. Next, samples were analyzed using a bottom-up approach. Results obtained were showing many oxidation products, such as amino side chain oxidation, however no AGEs/ALEs were identified. Thus, a new approach was adopted, including the incubation of plasma directly with RCS, such as HNE, MDA and ACR. This resulted in the formation of AGEs/ALEs in plasma samples, however, they could not be retained by the VC1 domain. Instead of using the VC1 technique to enrich AGEs/ALEs from biological samples, other variables throughout the experimental set-up were optimized. Previously, peptides were analyzed using the Orbitrap LTQ XL, a very powerful instrument. Nonetheless, the newest generation of tribrid MS offers even higher resolution, and it increases protein coverage due to parallel isolation and detection, and faster analyzers. Moreover, we focused on AGEs/ALEs from HSA and using NaBH4 to reduce and stabilize adducts throughout the analysis. This new approach permitted us to identify many AGEs/ALEs in both healthy human plasma samples, but also AGEs/ALEs only present in heart failure samples. Glycation on lysine residues was the main modification identified, present in both healthy and heart failure plasma samples. Important is the HNE Michael adduct, specifically identified in only heart failure samples. Moreover, the importance of stabilizing adducts is underlined by the fact that the acrolein Michael adduct could only be identified after reduction with NaBH4. Development of a cellular assay to determine pro-inflammatory activity of RAGE binders Another part of this project was focused on elucidating whether AGEs/ALEs induce an inflammatory response in cells. For this purpose, a collaboration was started with the Laboratory of Vascular Biology and Regenerative Medicine, Centro Cardiologico Monzino. Using a rat epithelial lung cell line overexpressing RAGE, and a control cell line not expressing RAGE, it could be detected if AGEs/ALEs exhibit an effect by binding to RAGE. Moreover, both cell lines were transfected with a Nf-κB reporter gene allowing us a fast and sensitive method for determining if binding of RAGE induces a down-stream signaling pathway. This system implies a firefly luciferase gene downstream from the Nf-κB gene. When the Nf-κB pathway is activated, independently from RAGE, it produces the firefly luciferase enzyme. After adding a luciferin substrate, firefly luciferase is able to convert this substrate into another substrate with light as by-product, which can be measured by a luminometer. IL-1α was used as a positive control, since it induces a strong inflammatory response through Nf-κB. Moreover, known ligands of RAGE able to activate the Nf-κB pathway, were used to validate the cellular experiment, including HSA modified with fructose (AGE), and HMGB1. Results show that Nf-κB is already increased in untreated cells with RAGE and that AGEs induce the Nf-κB pathway independently from RAGE. Moreover, the difference between control and RAGE cells is not significantly increased in the presence of HMGB1 compared to untreated. However, the positive control seemed to induce a much stronger activity in cells with RAGE. Overall, this cellular assay is good for assessing pro-inflammatory activity, however, it is not optimized yet for distinguishing a RAGE-dependent mechanism. CONCLUSION In summary, by using an integrated MS (intact protein and bottom-up approach) and computational approach we have found that some ALEs generated from lipid peroxidation RCS are RAGE binders. We have also found the basic features that ALEs from HNE, MDA and ACR must have to be a RAGE binder: 1) the covalent adducts should greatly reduce or abolish the basicity of the target amino acid, 2) the basic amino acid should be at the center of a set of carboxylic acids which, once the residue is modified, become available to freely contact the RAGE positive residues. Next step was to use the VC1 technique to enrich AGEs/ALEs in biological samples. First, oxidized human plasma was used, however, using the Orbitrap LTQ XL, it was not sufficient to identify AGEs/ALEs. Therefore, analysis was moved to a higher resolution mass spectrometer, which allowed us to identify AGEs/ALEs in plasma samples of heart failure patients, showing the powerfulness of this new generation MS. Important was to understand whether ALEs could induce pro-inflammatory activity through RAGE, since we showed that ALEs are RAGE binders. Unfortunately, the cellular assay that was set up is efficiently in determining Nf-κB dependent pro-inflammatory activity, but not if it is RAGE dependent.

Thesis (Ph. D.)--University of California, San Diego, 2008.
Title from first page of PDF file (viewed September 3, 2008). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (p. 104-126).

Die Interaktion von S100-Proteinen mit dem Rezeptor für advanced glycation endproducts (RAGE) wird als hoch relevant bei der Entstehung, Manifestation und Progression verschiedener entzündlicher Erkrankungen sowie bei der Tumorigenese gewertet. Das tiefergehende Verständnis der Interaktion von S100-Proteinen mit RAGE in vivo stellt eine wissenschaftliche Herausforderung dar und ist ein Ansatz für therapeutische Interventionen. Darüber hinaus stellen Untersuchungen zum Metabolismus von extrazellulär zirkulierenden S100-Proteinen in vivo einen vielversprechenden Forschungsansatz zur Analyse von S100-Protein-assoziierten Erkrankungen dar. Die einzigartigen Eigenschaften der Positronen-Emissions-Tomographie (PET) als nicht-invasives bildgebendes Verfahren erlauben die Darstellung und quantitative Erfassung biochemischer Prozesse mit der Möglichkeit zelluläre und molekulare Reaktionswege aufzuzeigen sowie in vivo-Mechanismen von Krankheiten im Kontext eines physiologischen Umfeldes darzulegen. Ziel der vorliegenden Arbeit war es, Fluor-18-markierte S100-Proteine (18F-S100) herzustellen, diese biochemisch, radiochemisch und radiopharmakologisch zu charakterisieren und deren Metabolismus und Interaktion mit RAGE in vivo mittels Kleintier-PET am Tiermodell zu untersuchen. Es wurden die mit RAGE interagierenden S100-Proteine S100A1, S100A12 und S100B in biologisch funktioneller Form hergestellt. Dazu wurden die entsprechenden S100-Gene in den prokaryotischen Expressionsvektor pGEX-6P-1 kloniert. Mit diesen Konstrukten wurden E. coli-Zellen transformiert, aus denen nachfolgend die S100-Proteine isoliert und gereinigt werden konnten. Es konnte eine Reinigung unter nativen, milden Bedingungen etabliert werden, die es ermöglichte, S100A1, S100A12 und S100B in biologisch aktiver Form und in hohen Reinheitsgraden (> 95%) für die nachfolgenden Experimente bereitzustellen. Diese S100-Proteine wurden über den 18F-tragenden Aktivester N-Succinimidyl-4-[18F]fluorbenzoesäure ([18F]SFB) radioaktiv markiert und charakterisiert. Dabei konnte sichergestellt werden, dass die 18F-S100-Proteine in vitro und in vivo stabil sind. Weiterhin konnte nachgewiesen werden, dass die radioaktive Markierung keine Beeinträchtigung auf die biologische Funktionalität der S100-Proteine hat. Dies wurde anhand von sRAGE-Bindungsuntersuchungen sowie Zell-Interaktionsuntersuchungen an konfluenten Endothelzellen (HAEC) und an zu Makrophagen differenzierten THP-1-Zellen (THP-1-Makrophagen) verifiziert. Für die Untersuchung der RAGE-Bindung war die Produktion des löslichen sRAGE bzw. die Generation von flRAGE-berexprimierenden Zellen erforderlich. Beide Konstrukte wurden in geeigneten Zellsystemen exprimiert und das sRAGE-Protein wurde in biologisch aktiver Form synthetisiert und gereinigt (Reinheitsgrad > 97%). Die 18F-S100-Bindung an THP-1-Makrophagen und HAEC wurde in Gegenwart von glykierten LDL (glykLDL) sowie sRAGE signifikant inhibiert, was auf eine RAGE-Interaktion hinweist. Weiterhin konnten durch den Einsatz von Scavenger-Rezeptor-Liganden, wie z. B. Maleinanhydrid-modifiziertes BSA (malBSA) bzw. von Lektinen inhibierende Effekte erzielt werden. Dies ist ein Indiz für die 18F-S100-Interaktion mit Scavenger-Rezeptoren und Glykokonjugaten an der Zelloberfläche. Durch die Untersuchungen mittels konfokaler Laserscanning-Mikroskopie an THP-1-Makrophagen wurde eine Zellaufnahme des Fluoreszein-markierten S100A12 festgestellt. Weiterhin konnten Kolokalisationen mit Lektinen detektiert werden. Das metabolische Schicksal extrazellulär zirkulierender 18F-S100-Proteine in vivo wurde mit Hilfe dynamischer PET-Untersuchungen bzw. anhand von Bioverteilungs-Untersuchungen in männlichen Wistar-Ratten analysiert. Die Hauptakkumulation der Radioaktivität wurde in der Leber und in den Nieren detektiert. In diesen Organen findet der Metabolismus bzw. die glomeruläre Filtration der 18F-S100-Proteine statt. In den Untersuchungen zur Genexpression mittels Echtzeit-PCR sowie im immunchemischen Proteinnachweis am Western Blot wurde eine hohe Expression und Proteinbiosynthese des RAGE in der Lunge ermittelt. Die Lunge eignet sich daher als „Referenz“-Organ für eine funktionelle in vivo-Charakterisierung von RAGE mit 18FS100-Proteinen. Bei den durchgeführten PET-Untersuchungen konnte eine temporäre 18F-S100-Interaktion mit dem Lungengewebe festgestellt werden. Die Retention des 18FS100A12 in der Lunge wurde in Gegenwart von sRAGE inhibiert. Dies ist ein Hinweis dafür, dass 18F-S100-Proteine auch in vivo an RAGE binden können. Die Radioaktivitäts-Akkumulation in den Organen Leber und Milz, die eine Vielzahl von sessilen Makrophagen aufweisen, wurde durch die Applikation von malBSA inhibiert. Dies ist ein Indiz dafür, dass 18F-S100-Proteine in vivo mit Scavenger-Rezeptoren interagieren können. Die vorliegende Arbeit liefert deutliche Hinweise darauf, dass RAGE nicht der alleinige Rezeptor für 18F-S100-Proteine ist. Der Einsatz von 18F-S100-Proteinen als experimentelles Werkzeug in dynamischen PET-Untersuchungen birgt das Potential einer Charakterisierung von S100-Protein-assoziierten, pathophysiologischen Prozessen
Members of the S100 family of EF-hand calcium binding proteins play important regulatory roles not only within cells but also exert effects in a cytokine-like manner on definite target cells once released into extracellular space or circulating blood. Accordingly, increased levels of S100 proteins in the circulating blood have been associated with a number of disease states, e.g., diabetes, cancer, and various inflammatory disorders. As the best known target protein of extracellular S100 proteins, the receptor for advanced glycation endproducts (RAGE) is of significant importance. However, the role of extracellular S100 proteins during etiology, progression, and manifestation of inflammatory disorders still is poorly understood. One reason for this is the shortage of sensitive methods for direct assessment of the metabolic fate of circulating S100 proteins and, on the other hand, measurement of functional expression of extracellular targets of S100 proteins, e.g., RAGE in vivo. In this line, small animal PET provides a valuable tool for noninvasive imaging of physiological processes and interactions like plasma or vascular retention, tissue-specific receptor binding, accumulation or elimination in vivo. To address this question, human S100 proteins were cloned in the bacterial expression vector pGEX-6P-1, expressed in E. coli BL21, and purified by affinity chromatography and anion exchange chromatography. Purified S100A1, S100B and S100A12 proteins were then radiolabeled with the positron emitter fluorine-18 (18F) by N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB). Radiolabeling of S100 proteins resulted in radiochemical yields of 3-10% (corrected for decay) and effective specific radioactivities of 1 GBq/µmol, respectively. For investigations about RAGE binding soluble RAGE (sRAGE) was expressed and purified using pSecTag2B. A radioligand binding assay confirmed specific binding of 18F-S100A12, 18F-S100A1, and 18F-S100B to immobilized sRAGE, also showing an order of affinity with S100A12 > S100A1 > S100B. These results indicate that radioactive labelling of S100 proteins did not affect their overall affinity to RAGE. Cellular association studies in human THP-1 macrophages and human aortic endothelial cells (HAEC) showed specific binding of all 18F-S100 proteins to the non-internalizing RAGE as confirmed by inhibitory effects exerted either by other RAGE ligands, e.g., glycated LDL, or by soluble RAGE. Of interest, 18F-S100 proteins were also shown to interact with other putative binding sites, e.g. scavenger receptors as well as proteoglycans. In this line, uptake of 18F-S100 proteins in THP-1 and HAEC could be inhibited by various scavenger receptor ligands, in particular by maleylated BSA as well as by lectines (e.g. ConA and SBA). Confocal laser scanning microscopy analysis showed a major part of the fluoresceinated S100A12 bound to the surface of THP-1 macrophages. Beyond this, uptake of S100A12 could be determined indicating an interaction of S100A12 with both non-internalizing, e.g., RAGE, and internalizing receptors, e.g. scavenger receptors. By evaluation of the relative contribution of 18F-S100A12 association to RAGE-overexpressed CHO cells (using pIres2-AcGFP1), 18F-S100A12 showed a significantly higher association to CHO-RAGE cells compared with CHO-mock cells. Based on these findings and due to their crucial role in inflammatory disorders the metabolic fate of S100 proteins was further investigated in dynamic small animal Positron emission tomography (PET) studies as well as in biodistribution studies in Wistar rats in vivo. For interpretation of in vivo investigations in rats, expression of RAGE was analyzed by quantitative real time RT-PCR as well as western blotting in various organs. Lung tissue expressed the highest level of RAGE protein compared to the other tissues. PET studies in rats revealed a comparatively long mean residence time of circulating 18F-S100 proteins. A major contributor to this phenomenon seems to be a sustained temporary interaction with tissues overexpressing RAGE, e.g., the lung. On the other hand, renal clearance of 18F-S100 via glomerular filtration is a major elimination pathway. However, scavenger receptor-mediated pathways in the liver, the spleen and, to a minor extent, in the kidneys, also seem to contribute to the overall clearance. The presence of sRAGE revealed a decreased retention of 18F-S100A12 in the lung, indicating in vivo binding to RAGE. In vivo blocking studies using maleylated BSA demonstrated a strong inhibition of putative binding sites in rat tissues enriched in cells expressing scavenger receptors like liver and spleen. In conclusion, 18F-labeling of S100 proteins and the use of small animal PET provide a valuable tool to discriminate the kinetics and the metabolic fate of S100 proteins in vivo. Furthermore, the results strongly suggest an involvement of other putative receptors beside RAGE in distribution, tissue association and elimination of circulating proinflammatory S100 proteins. Moreover, the approach provides novel probes for imaging of functional expression of RAGE and scavenger receptors in peripheral inflammatory compartments

Das S100A4-Protein ist für die Manifestierung eines metastatischen Phänotyps bei vielen Tumorarten von enormer Bedeutung. Die Aufklärung der zugrunde liegenden Mechanismen und der Interaktionspartner von S100A4 stellt daher einen vielsprechenden Forschungsansatz dar, um neue Erkenntnisse über das Verhalten von Tumorzellen während des Metastasierungsprozesses zu erhalten. Darauf aufbauend können neue Ansatzpunkte für die Therapie metastasierender Krebserkrankungen gewonnen werden. In dieser Hinsicht ist das bisher einer Behandlung kaum zugängliche maligne Melanom als besonders aggressiver und frühzeitig metastasierender Tumor ein ideales Modell zur Aufklärung der zellulären und molekularen Prozesse, über die S100A4 seine Metastasen-fördernden Wirkungen ausübt. Das Ziel der vorliegenden Arbeit war die biochemische und radiopharmakologische Charakterisierung der S100A4-RAGE-Interaktion sowie die Untersuchung der Beteiligung von S100A4 an Prozessen der Metastasierungskaskade in vitro und in vivo. Dies erforderte die Herstellung von rekombinantem S100A4-Protein und die Generierung von stabil mit S100A4-transfizierten Melanomzellen, die damit eine heraufregulierte S100A4-Proteinbiosynthese aufweisen. Die Gewinnung von rekombinantem S100A4 in biologisch funktioneller Form unter Verwendung eines prokaryotischen Expressionssystems erfolgte mit einem Reinheitsgrad von ca. 92%. Das rekombinante S100A4-Protein wurde mit dem Aktivester N-Succinimidyl-4-[18F]fluorbenzoat radioaktiv markiert und charakterisiert. Es wurde die Interaktion zwischen S100A4 bzw. 18F-markiertem S100A4 und der löslichen RAGE-Isoform sRAGE mit einer moderaten Bindungsaffinität im µM-Bereich nachgewiesen. Des Weiteren erfolgte erstmals die Analyse der radiopharmakologischen Eigenschaften von 18F-S100A4 mittels Untersuchungen zur zellulären Assoziation sowie zur metabolischen Stabilität, Bioverteilung und zu In-vivo-Interaktionen mittels Kleintier-Positronen-Emissions-Tomographie in der Ratte. Die In-vitro-Experimente wurden an Endothelzellen (HAEC) und an stabil mit RAGE-transfizierten A375-, A375-mock bzw. nicht transfizierten A375-Melanomzellen durchgeführt. Die A375-hRAGE-Zellen zeigten eine deutlich heraufregulierte RAGE-Proteinbiosynthese während die Endothelzellen eine vergleichsweise geringe intrazelluläre RAGE-Proteinkonzentration aufwiesen. Bei den Melanomzellen kann aufgrund der höheren Assoziation von 18F-S100A4 an A375-hRAGE-Zellen auf eine selektive Bindung von 18F S100A4 an RAGE-Rezeptoren auf der Zelloberfläche geschlossen werden. Die Assoziation von 18F S100A4 an Endothelzellen war bei 37°C in Gegenwart von nicht markiertem rekombinantem S100A4 signifikant vermindert, dementsprechend findet eine spezifische Interaktion von 18F-S100A4 mit Zelloberflächenrezeptoren der Endothelzellen statt. Dieses Ergebnis und die insgesamt höhere Bindung von 18F S100A4 an Endothelzellen im Vergleich zur Assoziation an Melanomzellen lassen neben RAGE noch andere Rezeptoren wie z. B. internalisierende Scavenger-Rezeptoren vermuten. Die In-vivo-Stabilitätsuntersuchungen verdeutlichen einen proteolytischen Abbau von 18F S100A4, allerdings belegen das Vorhandensein von 67% intaktem 18F-S100A4-Protein nach einer Stunde, die Stabilität von 18F-S100A4 in vivo. Die Bioverteilungs- bzw. PET-Untersuchungen zeigen eine schnelle, innerhalb weniger Minuten stattfindende hohe Akkumulation in den Nieren und verdeutlichen somit die renale Ausscheidung von 18F S100A4. Die maßgeblichen Anreicherungen in Milz, Leber, Blut, Lunge und Nebennieren lassen Interaktionen mit Oberflächenrezeptoren dieser Gewebe erkennen. Die temporäre Retention von 18F-S100A4 in der Lunge, dem Hauptsyntheseorgan von RAGE, und die verminderte 18F-S100A4-Akkumulation in Gegenwart des spezifischen RAGE-Liganden glykLDL ist ein Hinweis dafür, dass S100A4 in vivo in der Lunge an RAGE bindet. Die Aktivitätsanreicherungen in Milz, Leber und Nebenniere deuten aufgrund der geringeren RAGE-Synthese in diesen Organen auf die Interaktion von 18F-S100A4 mit anderen Zelloberflächenrezeptoren z. B. aus der Familie der Scavenger-Rezeptoren hin. Die Beteiligung von S100A4 an Metastasierungsprozessen des malignen Melanoms wurde an stabil mit S100A4-transfizierten A375-Melanomzellen, die eine Heraufregulierung der humanen bzw. murinen S100A4-Proteinbiosynthese im Vergleich zu A375-mock- (Vektor-Kontrolle) und nicht-transfizierten A375-Zellen zeigen, untersucht. Die A375-hS100A4-Zellen sezernierten zudem eine signifikant höhere S100A4-Proteinkonzentration in das umgebende Zellkulturmedium im Vergleich zu den Kontrollen. In dieser Hinsicht konnte bei den A375-hS100A4-Zellen, vermutlich aufgrund der höheren extrazellulären S100A4-Konzentration, eine gesteigerte Proliferations-, Motilitäts-, Migrations- und Invasionsrate gegenüber den A375-mock- und A375-Zellen nachgewiesen werden. In diesem Zusammenhang stehen ebenso die gesteigerte RAGE-Proteinbiosynthese und die signifikant höhere Aktivität des Transkriptionsfaktors NF-κB bei A375-Zellen nach 24-stündiger Inkubation mit Kulturmedium der A375-hS100A4-Zellen. Demnach wirkt vermutlich das extrazelluläre S100A4-Protein als autokriner bzw. parakriner Regulator von RAGE und NF κB. Die subkutane Injektion der A375- und stabil transfizierten A375-Melanomzellen in Nacktmäuse führte zur Entwicklung subkutaner Tumore an der Injektionsstelle. Bereits zwei Wochen nach der Injektion etablierten die A375-hS100A4-Zellen die signifikant größeren Tumore im Vergleich zu den A375-mS100A4-, A375-mock und A375-Zellen. Nach Injektion der Zellen in die Schwanzvene der Nacktmäuse konnte keine Entwicklung von Metastasen im Tierkörper festgestellt werden. IN DER VORLIEGENDEN ARBEIT WURDE NACHGEWIESEN: • RAGE ist ein Rezeptor für das S100A4-Protein. Allerdings gibt es eindeutige Hinweise für weitere S100A4-Zielproteine an der Zelloberfläche. • Die bedeutende Rolle von extrazellulärem S100A4 bei wichtigen zellulären Metastasierungsprozessen sowie bei der Aktivierung von Signalproteinen wie NF-κB und RAGE beim malignen Melanom. Die weitere Aufklärung der S100A4-spezifischen Signalkaskaden und Rezeptoren bei metastasierenden Tumorerkrankungen sowie die Charakterisierung von S100A4 als klinischen Parameter bei Patienten mit malignem Melanom stellen hoch interessante Aspekte in der Krebsforschung dar.

Le diabète est un important problème de santé publique en raison des complications vasculaires qu’il engendre. Il a été montré que la metformine (met), biguanide très utilisé dans le traitement du diabète de type 2, possède des effets vasculoprotecteurs indépendants de son effet antihyperglycémiant. Dans ce travail nous avons recherché son effet sur l’activité de la protéine kinase C (PKC) et de la polyADP-ribose polymérase (PARP), deux voies de signalisation majeures activées par l’hyperglycémie et décrites comme étant très impliquées dans le développement des complications vasculaires des patients diabétiques. Nous avons également étudié l’effet de la met sur l’expression du récepteur (RAGE) des produits de glycation avancée et du récepteur des LDL oxydés (LOX-1), marqueurs du dysfonctionnement endothélial artériel liés à la macroangiopathie diabétique. Nos résultats nous ont permis de proposer un mécanisme d’action de la metformine dont le site initial se situe au niveau membranaire
Type 2 diabetes is an important public health problem due to vascular complications caused by chronic hyperglycemia. Hyperglycemia has been shown to induce intracellular formation of reactive oxygen species and causes oxidative stress leading to vascular dysfunction. Metformin (met) is a widely used drug for the management of type 2 diabetes. Several studies showed that met improves vascular function of diabetic patients but its exact mechanism of action remains unclear. We investigate in this study the effect of met on the protein kinase C (PKC) and polyADP-ribose polymerase (PARP) pathways involved in diabetic vascular complications. We also explored whether met could modulate the redox-sensible expression of advanced glycation end products receptor (RAGE) and lectin-like oxidized receptor 1 (LOX-1). Taken together our results suggests that met acts at the membrane level and we proposed a unifying mechanism by which metformin improves diabetic vascular endothelial functions

RAGE is a multi-ligand pattern recognition receptor. RAGE can bind several damage associated molecular pattern proteins. RAGE- ligand interaction is pathophysiologically relevant to several major diseases including diabetes and certain cancers. RAGE inhibition has been reported to reduce morbidity in these disease states. However, to design better RAGE inhibitors it is necessary to understand the structural basis behind the RAGE-ligand interaction and currently this is not well understood. This thesis focuses on understanding the interaction of RAGE with two of its ligands; AGEs and S100B. AGEs are highly heterogeneous and are formed as a result of non-enzymatic glycation. A panel of AGEs were characterized in terms of their side chain modifications, thermal stability, secondary structure, aggregation and surface charge. These glycation induced changes were then correlated to RAGE binding. Building on these results the role of AGE-RAGE interaction in pancreatic cancer cell proliferation and migration was determined. Ribose modified BSA induced ROS formation, which then triggered NF-?B upregulation via RAGE induced ROS signaling. Ribose BSA increased pancreatic cell proliferation and migration. Anti-RAGE antibodies and RAGE inhibitors prevented AGE induced cellular effects. The role of ribose modified BSA was also determined in macrophage activation and pro-inflammatory cytokine release. Rapid internalization was observed of the ribose-BSA and confocal imaging revealed the internalization of the AGE compound into the lysosomes which lead to the ROS production, NF-?B activation and pro-inflammatory cytokine release in a RAGE independent signaling mechanism. Finally, the role of tryptophan residues of the V domain in domain stability and S100B binding was determined. We have generated single, double and triple tryptophan mutants of the V domain by site directed mutagenesis. The effect of Trp residues in the domain stability could not elucidated as no change was observed in the secondary structure of the mutants when compared to the wild type suggesting the plasticity of the V-domain. The fluorescence emission and life time properties of each Trp residue was determined. Our binding assays of the Trp Ala mutants indicate tighter binding of the S100B to the mutants. The S100-RAGE peptide structures suggest multi modal interaction of S100B-RAGE interaction.

The receptor for advanced glycation endproducts (RAGE) is a 35-kDa polypeptide of the immunogloblin superfamily that has been implicated as a mediator of both acute and chronic vascular inflammation. RAGE has also recently been implicated in the pathology of pulmonary hypertension (PH): a rare, progressive disease of the small pulmonary arteries characterised by pulmonary vascular remodelling, thrombosis, vasoconstriction and increased pulmonary vascular resistance. A ligand for RAGE, the calcium binding protein MTS1/S100A4, is expressed in occlusive vascular lesions of patients with advanced PH. MTS1/S100A4 is upregulated and secreted by pulmonary arterial smooth muscle cells (PASMCs) in vitro on activation of the 5HT1b receptor and 5HT transporter (5HTT). Additionally, the proliferative effect of 5HT on these cells, which is mediated by 5HT1b and 5HTT, may be inhibited by antagonism of RAGE or reduced bioavailability of MTS1/S100A4. These data suggest that MTS1/S100A4, through its action at RAGE, is a key mediator of 5HT-induced hPASMC proliferation. Transgenic mice overexpressing MTS1/S100A4 are observed to develop obliterative pulmonary vascular disease and possess increased right ventricular pressure at baseline and after hypoxia when compared to wildtype mice (WT). These increases occur in the absence of an increase in pulmonary vascular remodelling suggesting that MTS1/S100A4 overexpression is associated with some other structural or functional change in the pulmonary circulation. We sought to further our understanding of the role of RAGE in pulmonary hypertension through treatment with a small molecule inhibitor of monocyte chemoattractant protein 1(MCP-1), a marker of downstream of RAGE rage activation; through further characterisation of the MTS1/S100A4 mouse in a chronic hypoxic model of PAH; and through treatment with soluble RAGE (sRAGE) to reduce RAGE ligand bioavailability in vivo. In each case systolic right ventricular pressure (sRVP), right ventricular hypertrophy (RVH) and pulmonary vascular remodelling were measured in normoxic conditions or after a two week chronic hypoxia challenge to induce PH. These in vivo experiments were supplemented with functional studies in isolated intrapulmonary arteries to assess vascular reactivity and vascular elastance as well as studies of pulmonary fibroblast proliferation in vitro. Treatment with the MCP-1 synthesis inhibitor Bindarit produced no detectable effects upon the pulmonary response of mice to chronic hypoxia, though this study may have been hampered by difficulties with the methylcellulose vehicle. MCP-1 produced no degree of proliferation in pulmonary fibroblasts and neither augmented nor inhibited proliferation induced by 5HT. We found little evidence for the exacerbation of PH in MTS1/S100A4 mice in normoxia, hypoxia or after 4 weeks of normoxic recovery. Mean RVP was elevated above that in WT mice exposed to hypoxia. However, MTS1/S100A4 mice appeared protected against hypoxia-induced vascular remodelling and decreases in vascular elastance. No other significant differences in sRVP, RVH or remodelling were observed between strains. Vessels isolated from MTS1/S100A4 mice tended towards an enhanced contractile response to 5HT in normoxia compared with vessels in WT mice but were also more sensitive to the nitric oxide donor SNP. These differences in vasoreactivity were largely abolished by exposure to hypoxia. Treatment with soluble RAGE (sRAGE) to reduce RAGE ligand bioavailability produced a significant reduction in sRVP after hypoxia in comparison to vehicle-dosed mice -possibly associated with the prevention of a hypoxia-induced decrease in proximal vascular elastance. However, no benefit upon the development of remodelling or the extent of RVH was observed. Vessels isolated from mice treated with sRAGE and challenged with hypoxia showed a marked increase in contractility. Further work demonstrated that sRAGE produces a small, slowly developing contraction in isolated vessels and that the maximal force of contraction to 5HT was markedly augmented in the presence of sRAGE. Finally, treatment with sRAGE did not inhibit fibroblast proliferation in vitro as induced by 5HT but was observed to cause a small degree of proliferation alone and to augment hypoxia-induced proliferation. In summary, we have reported a number of seemingly contradictory findings associated with RAGE in pulmonary hypertension. Treatment with sRAGE produced a beneficial reduction in hypoxia-induced PH associated with protection against decreased proximal vascular elastance but produced no change in hypoxia-induced RVH or remodelling as well as greatly increasing vascular contractility. MTS1/S100A4 mice show some evidence of deleterious changes to the pulmonary circulation, but these may be offset by beneficial compensatory mechanisms such as increased sensitivity to nitric oxide and protection against vascular remodelling. MTS1/S100A4 stimulates smooth muscle cell proliferation suggesting that it may involved pulmonary vascular remodelling. However, inhibition of RAGE was observed to enhance fibroblast proliferation in response to hypoxia here. Fibroblasts are important regulators of SMC proliferation in vivo. These findings therefore suggest a more complicated relationship between RAGE, its ligands and the remodelling process. Since both MTS1/S100A4 overexpression and sRAGE treatment in vivo produced findings which are difficult to reconcile using the currently employed techniques, it is clear that furthering our understanding of RAGE will require study with greater focus upon the interaction of different cell types in the pulmonary vasculature and the manner in which the disturbance of this may lead to alterations in the physical and physiological properties of the pulmonary circulation.

The immunoglobulin-like, transmembrane Advanced Glycation End product (AGE) Receptor (RAGE) is a pattern recognition receptor implicated in the transduction of pro-inflammatory signalling and processes. Over the past decade a substantial body of evidence has accrued implicating RAGE in the pathogenesis of several chronic inflammatory and vascular diseases such as diabetes, rheumatoid arthritis, amyloidosis, atherosclerosis and renal failure. More recently RAGE has been linked to cancer progression, possibly through its role in the inflammatory process. AGE products have been shown to exert their intracellular effects through ligation of their cognate receptor RAGE and the subsequent transactivation of NFKB signalling in several cellular contexts. Polycystic Ovary Syndrome (PCOS) is a reproductive endocrine disorder characterized by hyperandrogenism, chronic anovulation and insulin resistance, thus increasing the risk of diabetes mellitus in these patients. Non-enzymatically glycated AGEs are formed at an accelerated rate and accumulate in tissues in conditions of high glucose and oxidative stress. Interestingly, young normoglycemic women with PCOS exhibit higher serum AGE levels and increased RAGE expression in poly-cystic ovaries. RAGE is also regulated through the activity of the estrogen receptor (ER). The natural cyclical expression of estrogen throughout the menstrual cycle is perturbed in endometriosis even post menopause, suggesting that RAGE could also be dysregulated. Finally PCOS has been implicated in increased risk to endometrial cancer progression as has uterine exposure to the selective estrogen receptor modulator Tamoxifen (TX) therefore it is plausible that RAGE has a function in this disease. Objectives: The principal aims of this thesis were to characterise RAGE expression for the first time in fertile and infertile endometriotic and PCO human endometrium, and to initiate RAGE characterisation in endometrium obtained from patients with endometrial hyperplasia and cancer. Secondly, this thesis endeavoured to elucidate the transcriptional mechanisms regulating RAGE in vitro response to 17beta-estradiol and AGEs which are elevated in endometriosis and PCOS pathology respectively, and in endometrial cancer. Methodology: This project employed the use of real time Polymerase Chain Reaction (RT-PCR), Chromatin Immunoprecipitation (ChIP), Immunohistochemistry (IHC) and western blotting (WB). Results: Immunohistochemistry and RT-PCR data revealed that basal RAGE expression was significantly greater in PCO and endometriotic endometrium when compared to fertile controls, and significantly elevated in two cancer patients. RAGE was also characterised in endometrial cell models in which it was shown to be modulated at the mRNA and protein level by AGE-HSA, 17beta-estradiol (E2) and its antagonist 4-hydroxytamoxifen. Moreover, we have shown that RAGE is modulated by two distinct pathways through the estrogen receptor (ER) and NFKB. Novel ChIP results confirmed the presence of p65 and ER-alpha on the RAGE promoter at non- classical Spl and Apl sites in response to AGEs, E2 and TX. Conclusions: The results in this thesis may implicate endometrial RAGE expression in the infertility evident in women with PCOS and endometriosis. Furthermore, recent evidence implicates RAGE in mediating inflammation-driven tumourigenesis. Thus, over-expression of endometrial RAGE in PCOS and endometriosis, and in patients receiving tamoxifen for breast cancer treatment may predispose these women to an elevated risk of cancer.

Overexpression of the Receptor for Advanced Glycation End Products (RAGE) has been implicated in multiple diseases, including several types of cancer. In different types of cancer, RAGE has been shown to promote cell survival by either autophagy or activation of the transcription factor NF-κB. Based on what is known about RAGE, we hypothesized that the RAGE/ligand interaction at the cell surface promotes pancreatic cancer and melanoma cell survival by both pathways, autophagy and NF-κB activation. To study the role of RAGE in pancreatic cancer resistance to chemotherapy, BxPC-3, MIA PaCa-2, PANC-1, and RAGE overexpressing PANC-1 FLR2 cell-lines were used. A significant decrease in cell viability was observed upon gemcitabine treatment with further significant reduction in cell viability upon combination of gemcitabine with the RAGE inhibitor IgG 2A11. In our studies we showed that RAGE plays a central role in pancreatic cancer cell resistance to gemcitabine by increasing autophagy. To test the importance of RAGE localization in mediating drug resistance, three melanoma cell-lines (WM115, WM266, and SK-MEL2) with their daughters, RAGE overexpressing cells (WM115-RAGE, WM266-RAGE, and SK-MEL2-RAGE) were used. Wild type cell-lines only expressed RAGE intracellularly while RAGE overexpressing cells expressed RAGE both at the cell surface and inside cells. We show in this study that only the cell surface RAGE is involved in melanoma resistance to dacarbazine. We next tested the effects of RAGE/RAGE ligand interaction at the cell surface in pancreatic tumor growth. We used two carcinoma cell-lines, PANC-1 and MIA PaCa-2, for this purpose. Both cell-lines were transiently transfected with a NF-κB/Luciferase reporter plasmid to test the effects of the interaction between RAGE and its ligands on the activation of the NF-κB signaling pathway. We observed higher NF-κB activity upon treatment with RAGE ligands (AGE, S100P, and S100A8/A9) compared to non-treated cells. Higher activity of NF-κB was coupled with a higher expression of cyclin D1 and lower expression of p53, NF-κB target genes.
Cobre grant "P20GM109024"

Advanced glycation end-products (AGEs) are the products of a non-enzymatic reaction that occurs in the blood between glucose and albumin. The receptor for advanced glycation end-products (RAGE) is a transmembrane protein primarily located in the endothelial cells of small vasculature which binds AGEs. When RAGE binds to its ligands, it activates a chronic inflammatory response in the genes. While this is most likely a natural immune response, in diseases that result in chronically high levels of AGEs in the blood, such as diabetes, the chronic inflammation can cause damage to the vasculature. This occurs by altering the microenvironment of the basal membrane in tissues where RAGE is expressed. Complications such as edema, retinopathy, nephropathy and cardiovascular disorders can result from this inflammation. Birds have an average fasting blood glucose level 4-5 times higher than that of a human being, making them an ideal animal model for studying adaptation to chronic high blood glucose levels. Additionally, they , do not suffer from these RAGE-related inflammatory disorders. This suggests that RAGE may not be present in birds. Tissue from Mourning Doves (Zenaida macroura) was examined for the presence of RAGE through antibody based protein identification techniques. Though not strongly conclusive, the evidence suggests that RAGE is not present in the small vasculature of Mourning Doves.

Acute lung injury (All) is a devastating pulmonary condition with no effective therapeutic. Uncontrolled inflammation and tissue destruction cause rapid deterioration of gas exchange and often result in permanently impaired lung function or mortality. The receptor for advanced glycation end products (RAGE) is a multiligand receptor highly expressed on pulmonary epithelial and endothelial cells. RAGE and its ligands have been implicated in inflammation in a variety of diseases, including ALL This project showed that the RAGE ligands HMGBl and calgranulin C were present in high concentrations in the airspace of patients with ALL These ligands drove RAGE-dependant cell activation through MAPK and NF-KB pathways in several in vitro pulmonary epithelial and endothelial cell models, as well as in a novel ex vivo murine pulmonary tissue model. This cell activation was combined with increases in apoptosis, IL-8 and matrix metalloproteinase concentrations in these cells as well as decreases in TIMP-l(tissue inhibitor of metalloproteinase) anti-protease protection. If mirrored in vivo, these results ) would cause increases in inflammatory cell infiltration and destruction of the lung tissue, worsening inflammation in ALL When used as a pre-treatment, soluble RAGE (sRAGE), which is thought to act as a scavenger of RAGE ligands, showed potent inhibition of cell activation and reduced inflammatory molecule release mediated by RAGE-ligands and by lavage fluid isolated from the lungs of patients with ALL RAGE ligands caused MAPK and NF-KB cell activation as well as increases in MIP-2 and KC inflammatory markers in ex vivo mouse lung tissue. These responses were reduced in RAGE-KO animal tissue treated the same way and through use of sRAGE. This evidence suggests that RAGE adds to the uncontrolled inflammation seen in All and that sRAGE has potential as an inhibitor of the inflammatory functions of RAGE.

Le vieillissement est défini par l’accumulation d’événements menant à une perte d’efficacité des fonctions des organes et à une augmentation de la probabilité de mourir avec le temps. Ce processus touche tout le règne animal et bien que sa vitesse varie largement entre les espèces, modifiant grandement la longévité, ses mécanismes sont quant à eux bien conservés. Chez l’humain, l’espérance de vie a continuellement progressé au cours du siècle dernier, s’accompagnant d’une augmentation de personnes atteintes de maladies liées à l’âge et dépendantes, devenant ainsi un problème majeur de société.La glycation est une réaction non-enzymatique menant à une interaction irréversible entre des composés carbonylés, tels que les sucres, avec des nucléophiles, comme la lysine ou l’arginine, produisant des produits de la glycation avancée (AGE). L’accumulation des AGE avec l’âge dans le corps suggère que ce processus est particulièrement impliqué dans le vieillissement. Cependant, le rôle de la consommation d’AGE alimentaires sur le vieillissement est beaucoup moins connu. Leur digestion implique d’importantes modifications structurelles et ceux-ci ne peuvent qu’avoir des effets indirects. Notre équipe a démontré qu’une consommation prolongée d’un régime enrichi en carboxymethyllysine (CML), un des AGE les plus abondants, induisait un vieillissement vasculaire accéléré chez des souris d’âge moyen. Cependant cet effet était complètement dépendant de l’expression du récepteur aux AGE, RAGE.RAGE est un récepteur multiligand et son activation est principalement définie par une réponse pro-inflammatoire auto-alimentée qui a été impliquée dans des maladies liées ou non à l’âge telles que les complications du diabète, des maladies cardiovasculaires, la maladie d’Alzheimer ou différents cancers. Étant donné le lien entre les AGE et RAGE et leur implication dans le vieillissement, nous émettons l’hypothèse que RAGE tient un rôle important dans le vieillissement physiologique et accéléré par les AGE. De plus, notre équipe a également démontré que la CML alimentaire s’accumulait principalement dans les reins chez la souris. Par conséquent, nous cherchons à déterminer si la CML alimentaire accélère également le vieillissement rénal chez les souris et si la suppression de RAGE empêche cet effet et a un impact sur le vieillissement normal.Des souris sauvages (WT) et RAGE-/- de 2 mois ont été nourries pendant 18 mois avec un régime contrôle ou enrichi en CML (200μg CML/gnourriture). La répartition de la CML a été déterminée par immunohistochimie et en HPLC-MS/MS. Le vieillissement du rein a été évalué en mesurant des marqueurs de sa fonction, de ses lésions, d’amylose ainsi que d’inflammation, d’oxydation et de vieillissement. Enfin, nous avons également évalué la fonction motrice chez de vieilles souris (~22 mois) en utilisant des tests de la locomotion.Bien que la CML s’accumulait dans les reins de souris nourries avec le régime enrichi en CML, celui-ci n’avait que peu d’effets sur les paramètres étudiés alors que les souris ne possédant pas RAGE étaient fortement protégées contre les lésions rénales liées au vieillissement, l’amylose sénile rénale et l’inflammation à bas bruit alors que des voies pro-longévité étaient renforcées. Nous montrons ensuite que certaines fonctions motrices des vieilles souris RAGE-/- pourraient être mieux conservées que chez les vieilles souris WT, supposant une sarcopénie moins important chez les souris RAGE-/-.L’impact conséquent de RAGE sur le vieillissement et sur l’inflammation chronique à bas bruit, associé à ses caractéristiques intrinsèques, suggèrent fortement que RAGE est un récepteur de reconnaissance de motifs moléculaires (PRR) et est une preuve de principe que « l’inflammaging » est un moteur important du vieillissement qui reste néanmoins modulable, génétiquement ou pharmacologiquement
Ageing is defined by the accumulation of events leading to a reduction in the efficacy of organ functions and an increased probability of death with time. This process affects all the animal kingdom and while the pace of ageing varies significantly among species, greatly affecting longevity, the mechanisms of ageing itself are widely conserved. In humans, as life expectancy at birth has been steadily increasing for over a century, the amount of people with age-related diseases and dependency has greatly increased and is becoming a major concern.Glycation is a non-enzymatic process leading to the irreversible interaction of carbonyl compounds, such as sugars, with nucleophiles, including lysine or arginine, forming advanced glycation end-products (AGEs). This process is thought to be involved in ageing as AGEs accumulate in the body with age. However, the role in ageing of consuming AGEs produced during cooking processes is much less understood. Digestion vastly modifies their structure and they can only have indirect an impact. Our group has shown that the long-term consumption of a diet enriched with carboxymethyllysine (CML), one of the most abundant AGEs, induced an accelerated vascular ageing in middle-aged mice. However, this effect was entirely dependent on the expression of the receptor for AGEs, RAGE.RAGE is a multiligand receptor and its activation is primarily characterised by a self-sustaining pro-inflammatory response which has been implicated in both age-related and age-independent disorders including complications of diabetes, cardiovascular diseases, Alzheimer’s disease or cancers. Given the relationship between AGEs and RAGE and their respective role in ageing or age-related disorders, it was hypothesized that RAGE has an important role in both physiological and AGE-accelerated ageing. In addition, our group has demonstrated that dietary CML mostly accumulates in mice kidneys, which age slower than vessels. Therefore, a key aim of this thesis was to investigate whether dietary CML also induces accelerated kidney ageing in older mice and whether the deletion of RAGE prevents this effect and has an impact on normal ageing.Two-month-old wild-type (WT) and RAGE-/- mice were fed a control or a CML-enriched diet (200μg CML/gfood) for 18 months. CML distribution was assessed by immunohistochemistry (IHC) and HPLC-MS/MS. Kidney ageing was assessed by measuring markers of its function, lesions and amyloidosis, as well as of inflammation, oxidation and ageing. In addition, motor function in old (~22 month-old) mice was also assessed using locomotion tests.Firstly, it was demonstrated that although CML accumulated in the kidneys of mice fed the CML-enriched diet, this diet had little effect upon the studied parameters while mice deprived of RAGE were largely protected against age-related renal lesions, renal senile amyloidosis and exhibited decreased inflammation and improved pro-longevity pathways. Thereafter, it was shown that some of old RAGE-/- mice motor functions might be better preserved than in old WT animals, suggesting a reduced sarcopenia in RAGE-/- mice.The significant impact of RAGE on ageing and on low-grade and chronic inflammation, associated with its intrinsic characteristic, strongly suggest that RAGE is a pattern recognition receptor and is a proof of principle that inflammaging is an important motor of ageing which may be modulated through genetic or possibly pharmacologic interventions

Expression of the Receptor for Advanced Glycation End Products (RAGE) and is upregulated in a several cancers. Based on published studies, we hypothesized that RAGE, when overexpressed in pancreatic cancer cells, will promote cell proliferation and migration. To study the role of RAGE in pancreatic cancer, we selected the human pancreatic cancer cell-line PANC-1, and stably transfected the cells with full length RAGE to generate model cell-lines that overexpress RAGE. We obtained two cell-lines PANC-1 FLR2 and PANC-1 FLR3 and examined the influence of RAGE on cellular properties. A significant increase in proliferation but a reduction in migratory abilities of PANC-1 FLR2 and PANC-1 FLR3 cells was observed. The increase in proliferation and reduction in migration was reverted upon knockdown of RAGE in PANC-1 FLR2 cells with siRNA specific for RAGE. The reduction in migration was supported by the reduced levels of vimentin and several integrins in RAGE transfected cells. Furthermore, we observed a downregulation in FAK, AKT, ERK1/2 and NF-κB activity. Growing evidence supports that RAGE is essential for pancreatic cancer progression. It has also been shown that RAGE facilitates pancreatic tumor cell survival by enhancing autophagy and inhibiting apoptosis. The goal of our study was to determine the effect of RAGE inhibition during gemcitabine chemotherapy on the growth of pancreatic tumor. Hence, we investigated the effect of RAGE inhibitors and their combination with gemcitabine in an orthotopic mouse model of pancreatic cancer using mouse pancreatic cancer cell-line KPC 5508. We used two RAGE inhibitors, an anti-RAGE monoclonal antibody (IgG2A11) and a small molecule RAGE inhibitor (FPS-ZM1). We observed a significant reduction in tumor weights of the mice treated with the combination of IgG2A11 and gemcitabine as compared to gemcitabine alone treated mice. The reduction in tumor growth was accompanied with increase in p62 levels (marker of autophagy) and increase in levels of cleaved PARP (marker of apoptosis). We also observed reduction in HMGB1 and phosphorylation levels of ERK1/2 in tumors from the group treated with the combination as compared to the gemcitabine alone treated group.
North Dakota State University. College of Health Professions
NIH Grant # P20 GM109024 from the National Institute of General Medicine

The Receptor for Advanced Glycation End products (RAGE) interacts with several classes of structurally unrelated ligands. The activation of RAGE by its ligands results in the cellular activation of several kinases and transcription factors including mitogen activated protein kinases (MAPKs) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) resulting in sustained inflammation, which is involved in pathologies such as diabetes, cancer, Alzheimer's disease, multiple sclerosis and other diseases associated with chronic inflammation. Current mouse models of human disease have shown that RAGE activity can be efficiently suppressed using either soluble RAGE (sRAGE) or anti-RAGE antibodies as inhibitors. Our goal was to generate new monoclonal antibodies against RAGE that can serve as diagnostic as wells as therapeutic tools in RAGE related pathologies. The chapters in this dissertation are a complete documentation of the development of these anti-RAGE antibodies. Additionally, an introductory review of antibodies, which includes structure and function, types of antibodies and production and basic understanding of RAGE and its ligands, has been provided to facilitate the understanding of the chapters. The first chapter details the development and characterization of anti-RAGE antibodies produced from hybridoma. The next chapter explores the effects of the generated antibodies to mammalian cells in in vitro settings and the final chapter applies the generated antibodies in vivo. During the course of this work, the antibodies developed showed binding to RAGE at nano-molar affinities which are comparable to the affinities of current antibodies used for therapeutic purposes, diagnostic and research purposes. We were also able to delineate that the possible mechanism of action of the antibodies is by preventing binding to RAGE. Lastly, we observed that one of the generated antibodies was able to reduce tumor growth in vivo in a melanoma xenograft mouse model.

The receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily. The result of RAGE-ligand interactions augments the proinflammatory mechanisms acting in chronic inflammatory diseases. RAGE recognises a wide range of ligands that have no apparent structural similarities. It is unclear what controls this promiscuity of RAGE. The extracellular domain of RAGE has two potential glycosylation sites. It is speculated that N-linked glycosylation may have significant impact on ligand recognition, especially of S100 calcium binding protein ligands. Two objectives of this thesis were to establish whether S100A9 acts as a ligand for RAGE and to investigate whether glycosylation of RAGE has any influence on ligand recognition. These were achieved by generating two forms of RAGE. HEK 293 cells were transfected to express full-length, membrane-bound RAGE or a secreted form comprising the extracellular domain of RAGE. Site-directed mutagenesis of RAGE showed that asparagine at position 25 is the pre-dominant N-linked glycosylation site. The carbohydrate added to asparagine 25 was further modified to a non-sialylated carboxylated N-linked glycan, specifically recognised by monoclonal antibody GB 3.1. Binding studies showed that different RAGE ligands have individual requirements for glycosylation of the receptor. Binding of AGE-modified AGE-BSA or of S100B to RAGE occured independent of N-linked glycosylation of the receptor. RAGE also binds the S100 protein, MRP-14 (S100A9). In contrast to AGE-BSA or S100B, the non-sialylated carboxylated N-glycan expressed on RAGE is crucial for binding to MRP-14. However, RAGE produced in tunicamycin containing medium and thus lacking N-linked glycosylation, shows strong binding to MRP-14. It was concluded that two forms of binding are involved: the first mechanism relies on the non-sialylated carboxylated N-glycan attached to RAGE and acts in a "tethering" fashion. The second mechanism involves a conformational change of RAGE, which results in exposure of a binding site(s) and a more conventional receptor-ligand interaction. Another objective for this thesis is to study the expression of RAGE and its alternatively spliced variants. PCR analysis has revealed several variants of RAGE that result from alternative splicing mechanisms. The variant proteins are soluble due to a lack of membrane localising sequence. PCR results confirmed the presence of transcripts encoding for spliced variants of RAGE in several tumour cell lines. Among these were transcripts that should encode a soluble form of sRAGE 2. Furthermore, it was shown that sRAGE 2 transcript can be present in forms that contain the ligand-binding V-domain of RAGE or that are N-truncated and lack the V-domain. This is the first report of a soluble, N-truncated sRAGE 2 variant. The results in this thesis add to our knowledge of RAGE biology. MRP-14 (S100A9) is identified as a new ligand. The control of MRP-14/RAGE interaction relies on N-linked glycosylation of the receptor and further modification of the carbohydrate. "Tethering" or stronger receptor-ligand interactions are suggested as mechanisms for controlling RAGE recognition of multiple ligands. Soluble RAGE variants that lack or contain V-domain binding regions, and hence sites for glycosylation were produced. These have the capacity to compete with membrane-bound receptor for available ligand. The control of the expression of soluble RAGE variants, in concert with the control of various modification to carbohydrate expressed on the receptor, adds a level of complexity to ligand specificity. This may ultimately result in different paradigms of the inflammatory process.

As humans, we inhabit an environment shared with many microorganisms, some of which are harmless or beneficial, and others which represent a threat to our health. A complex network of organs, cells and their protein products form our bodies’ immune system, tasked with detecting these potentially harmful agents and eliminating them. This same system also serves to detect changes in the healthy balance of normal functions in the body, and for repairing tissue damage caused by injury. Immune recognition of nucleic acids, DNA and RNA, is one way that the body detects invading pathogens and initiates tissue repair. A number of specialized receptor proteins have evolved to distinguish nucleic acids that represent “threats” from those involved in normal physiology. These proteins include members of the Toll-like receptor family and diverse types of cytosolic proteins, all of which reside within the confines of the cell. Few proteins on the cell surface have been clearly characterized to interact with nucleic acids in the extracellular environment. In this dissertation, I present collaborative work that identifies the receptor for advanced glycation end products (RAGE) as a cell surface receptor for nucleic acids and positions it as an important modulator of immune responses. Molecular dimers of RAGE interact with the sugar-phosphate backbones of nucleic acid ligands, allowing this receptor to recognize a variety of DNA and RNA molecules regardless of their nucleotide sequence. Expression of RAGE on cells promotes uptake of DNA and enhances subsequent responses that are dependent on the nucleic acid sensor Toll-like receptor 9. When mice deficient in RAGE are exposed to DNA in the lung, the predominant site of RAGE expression, they do not mount a typical early inflammatory response, suggesting that RAGE is important in generating immune responses to DNA in mammalian organisms. Further evidence suggests that RAGE interacts preferentially with multimolecular complexes that contain nucleic acids, and that these complexes may induce clustering of receptor dimers into larger multimeric structures. Taken together, the data reported here identify RAGE as an important cell surface receptor protein for nucleic acids, which is capable of modulating the intensity of immune responses to DNA and RNA. Understanding of and intervention in this recognition pathway hold therapeutic promise for diseases characterized by excessive responses to self nucleic acids, such as systemic lupus erythematosus, and for the pathology caused by chronic inflammatory responses to self and foreign nucleic acids.

Surgery necessitating cardiopulmonary bypass (snCPB) is associated with systemic inflammation which can be severe. Systemic inflammation is common in the critically ill, is associated with adverse outcome and currently has no specific therapy. Insight into the pathogenesis of systemic inflammation may lead to therapies. The receptor for advanced glycation end-products (RAGE) may represent a novel target for intervention. RAGE is a ubiquitous multi-ligand receptor that is up-regulated in the presence of its ligands. Initially characterised as a receptor for glycated proteins, it is also binds the S100 proteins and high mobility group box 1 (HMGB1); causing pro-inflammatory responses via NF-κB and the MAP kinases. RAGE inhibition has been associated with improved outcomes in animal models of infectious and sterile systemic inflammation. Of the snCPB patients assessed (n=2440) for relationships between age (associated with RAGE up-regulation) with systemic inflammation and clinical outcome, the oldest patients met more SIRS criteria in the first 1h and 24h following snCPB than those aged 40-80 y. This was accompanied by higher scores of organ dysfunction. Also, plasma levels of RAGE ligands and soluble RAGE increased (n=18-120) around surgery with pre-operative levels correlating with duration of intensive care. Leukocyte cell-surface and intracellular levels of RAGE were assessed and cell surface levels on neutrophils decreased following surgery, possibly contributing to the sRAGE levels in plasma. Cytokine release from whole blood increased following incubation with RAGE ligands, with a diminished effect on whole blood obtained after snCPB, suggesting leukocyte hypo-responsiveness. Finally, genotyping 8 single nucleotide polymorphisms in the RAGE, HMGB1 and S100A8 genes in 187 snCBP patients indicated statistically significant relationships to clinical outcomes such as impaired oxygenation and incidence of acute kidney injury. The findings from these investigations, inform understanding of the involvement of the RAGE axis in systemic inflammation.

This thesis will be organized into three chapters discussing the mechanism underlying the onset and progression of osteoarthritis (OA) in the temporomandibular joint (TMJ). Understanding the mechanism of OA development in the TMJ helps in understanding how OA progresses and how to treat this disease. The goal of this investigation is to examine the process of cartilage degeneration and OA biomarker expression in the TMJ to understand their role in TMJ OA onset and development.Chapter one covers mechanisms that are altered in TMJ OA during disease progression. Using animal models with different stressors such as mechanical disturbances, direct injury, and changes in the extracellular matrix composition revealed the role of the different mechanisms that are up-regulated and down regulated during cartilage destruction. Chapter two will cover a paper I wrote that introduces a novel non-invasive technique applied to mice, which induces an early onset of OA in the TMJ. I developed this technique with the aim to provide a new mouse model where the onset and progression of OA more closely mimic the natural TMJ OA progression in humans. The histopathological analysis of the cartilage demonstrates that onset of OA starts at 2 weeks after treatment induction and is aggravated by week eight. This data demonstrated the effectiveness of our technique in inducing OA in the TMJ. Chapter three will cover a second paper I wrote on the association of RAGE with the progression of OA in the TMJ of mice by using mice with and without RAGE expression. RAGE has been show to contribute to the progression of OA by releasing several pro-inflammatory and catalytic cytokines. Additionally, RAGE has been shown to modulate the expression of specific OA biomarkers, including HtrA-1, Mmp-13, and Tgf-β1 in knee cartilage. The objective of this study was to study the effect of knocking out RAGE on the expression of Mmp-1 3, HtrA-1, and Tgf-β1 in the TMJ. After histophatological and quantitative analysis of biomarkers expression, the results demonstrated for the first time that absence of RAGE expression in the TMJ provides a protective effect against development of TMJ OA in mice.

Background: L'invecchiamento è un inevitabile fattore di rischio in età avanzata che può influenzare l'insorgenza e la progressione di diverse malattie. Infatti, l'elevata incidenza di malattie cardiovascolari negli anziani è principalmente imputabile al fisiologico rimodellamento cardiaco associato ad un invecchiamento intrinseco. RAGE è un recettore capace di legare diverse molecole e coinvolto in molte malattie legate all'età. La sua isoforma solubile (sRAGE) agisce come un recettore decoy bloccando l'attivazione del recettore legato alla membrana, e suoi livelli circolanti sono stati trovati alterati in diverse patologie croniche ed acute. Il ruolo delle isoforme di RAGE durante l’invecchiamento e, in particolare, nell’invecchiamento cardiaco, non è mai stato studiato. Inoltre, la scoperta di biomarcatori affidabili in grado di valutare lo stato di salute individuale dei soggetti ha importanti applicazioni nel campo della prevenzione, della diagnosi e della gestione della malattia. In tale contesto, lo scopo di questo studio è stato quello di verificare se sRAGE sia un biomarcatore di invecchiamento e di rimodellamento cardiaco legato all’invecchiamento, e valutare il contributo delle isoforme RAGE nell’invecchiamento cardiaco. Risultati: È stato collezionato il siero di soggetti sani, di entrambi i sessi, di età compresa tra i 20 e i 92 anni ed i livelli di sRAGE sono stati valutati mediante ELISA. Abbiamo trovato una significativa diminuzione di sRAGE circolante nei maschi, mentre solo una tendenza nelle femmine. Di conseguenza, abbiamo osservato una forte correlazione di sRAGE con l'età cronologica nei soggetti maschi, ma non nei soggetti di sesso femminile. Topi maschi e femmine a diverse età (2.5-12-22 mesi, Giovani, adulti (MA) e Vecchi, rispettivamente) sono stati sottoposti a ecocardiografia 2D per determinare le dimensioni e la funzione del ventricolo sinistro (LV) durante l'invecchiamento. sRAGE serico diminuisce in maniera simile in entrambi i sessi tra il gruppo Giovani e il gruppo MA, e correla inversamente con le dimensioni e la funzione del LV, in particolare nei machi. Nessuna quantità rilevabile di RAGE è stata trovata nei lisati proteici del LV a tutte le età. Topi Rage-/- hanno mostrato un significativo aumento dei volumi e dei diametri del LV in diastole e in sistole, ed una concomitante diminuzione della frazione di eiezione (EF) e di accorciamento (FS), rispetto agli animali Rage+/+ di pari età durante l'invecchiamento con le più forti differenze presenti tra i gruppi MA. Inoltre, topi MA Rage-/- hanno mostrato la maggiore deposizione di collagene e l’aumento dell'espressione di geni marcatori di scompenso cardiaco (BNP e Ankrd1) rispetto alla controparte Rage+/+. Al contrario, nessuna differenza in termini di dimensioni dei cardiomiociti è stata osservata a qualsiasi età tra i due genotipi. Infine, l’analisi funzionale di annotazione del microarray, basata sull'interazione fra età-genotipo, ha rivelato che la mancanza cronica di RAGE influenza l'espressione di geni associati alla funzione contrattile, al processo di presentazione dell'antigene e dell'immunità adattativa, del pathway dell’insulina, della morte cellulare e dell’apoptosi. Abbiamo anche trovato una correlazione tra i volumi e i diametri del LV in diastole e in sistole e i geni differenzialmente espressi, i quali sono coinvolti in diversi processi come la contrazione muscolare, la fibrosi e la regolazione dell'apoptosi. Conclusioni: I nostri risultati indicano che sRAGE è un biomarcatore serico di invecchiamento sano e di rimodellamento cardiaco legato all'età, preferenzialmente nei maschi. L'assenza di RAGE aggrava l’avverso rimodellamento cardiaco legato all'età. Proponiamo che, tra le isoforme RAGE, sRAGE possa giocare un ruolo fondamentale nell’invecchiamento cardiaco.
Background: Aging is an unavoidable risk factor in later life that can influence the onset and progression of many diseases. In fact, the high incidence of cardiovascular diseases in the elderly is mainly attributable to cardiac remodelling associated to physiological intrinsic aging. RAGE is a multi-ligand receptor involved in many age-related disorders. Its soluble isoform (sRAGE) acts as a decoy receptor being able to block the activation of the membrane-bound receptor, and its circulation levels have been found altered in several chronic and acute pathologies. The role of RAGE isoforms in aging and, in particular, cardiac senescence has never been investigated. Moreover, the finding of reliable biomarkers able to assess individual health status of subjects has important applications in prevention, diagnosis, and disease management. In this context, the aim of this study was to ascertain whether sRAGE is a biomarker of aging and age-related cardiac remodelling, and evaluate the contribution of RAGE isoforms to cardiac aging. Results: Serum of male and female from 20 to 92 years old healthy subjects was collected and sRAGE levels were evaluated by ELISA. We found a significant decrease of circulating sRAGE in males while only a trend in females. Accordingly, we observed a strong correlation of sRAGE with chronological age in male but not in female subjects. Male and female mice at different age (2.5-12-22-months, Young, Middle Age (MA) and Old, respectively) undergone 2D-echocardiography to determine the left ventricle (LV) dimensions and function during aging. Serum sRAGE similarly declines from the Young to the MA group in both sexes, and inversely correlate with LV dimensions and function, preferentially in males. No detectable amount of RAGE protein was found in LV at all ages. Rage-/- mice displayed a significant increase of LV volumes and diameters in diastole and systole, and a concomitant decrease in ejection fraction (EF) and fractional shortening (FS), compared to age-matched wt animals during aging with the strongest differences present between the MA groups. Moreover, MA Rage-/- mice exhibited higher deposition of collagen and expression of heart failure marker genes (BNP and Ankrd1) in respect to the wt counterpart. Conversely, no differences in cardiomyocytes size were observed at any age between the two genotypes. Finally, microarray functional annotation analysis based on the interaction between age-genotype revealed that the chronic lack of RAGE affected the expression of genes associated to contractile fibre function, antigen presenting process and adaptive immunity, insulin pathway, cell death and apoptosis. We also found a correlation between LV volumes and diameters in diastole and systole and differentially expressed genes involved in several processes like muscle contraction, fibrosis, wound healing and regulation of apoptosis. Conclusions: Our results indicate that sRAGE is a serum biomarker of healthy aging and age-related cardiac remodeling, preferentially in males. The absence of RAGE in mice exacerbates adverse cardiac remodeling with age. We propose that, among RAGE isoforms, sRAGE may play a pivotal role in cardiac senescence.

High Mobility Group Box 1 (HMGB1) plays key roles in inflammatory diseases including cancer and diabetes. Intracellularly, HMGB1 binds DNA and assists transcription. Extracellularly, HMGB1 promotes inflammatory responses by binding to receptors such as the Receptor of Advanced Glycation End-products (RAGE). HMGB1 specifically self-associates in Surface Plasmon Resonance (SPR). The self-association is markedly influenced by ionic strength, redox, zinc ions, and pH. The role of HMGB1 self-association in binding DNA or RAGE is discussed.

Kohlenhydrate und Proteine gehören neben Wasser und Fetten zu den quantitativ bedeutendsten Grundbestandteilen biologischer Systeme und der Lebensmittel. Unter milden Bedingungen in lebenden Organismen oder unter thermischer Belastung bei der Lebensmittelverarbeitung können reduzierende Kohlenhydrate amin-katalysiert durch die Abspaltung von Wasser und Fragmentierungen des Kohlenstoffgerüsts abgebaut werden, wobei die noch reaktiveren 1,2-Dicarbonylverbindungen entstehen. Aus der Reaktion der N-α-Aminogruppe und funktioneller Gruppen der Seitenketten von Aminosäuren mit Kohlenhydraten bzw. 1,2-Dicarbonylverbindungen können stabile Endprodukte entstehen. In vivo können proteingebundene Maillard-Produkte (MRPs) aus der Reaktion mit Glucose (Amadori-Produkte) oder 1,2-Dicarbonylverbindungen (Advanced Glycation Endproducts: AGEs) entstehen. Beispielsweise ist das „N-terminale“ N-α-Fructosylderivat der β-Kette des Hämoglobins ein etablierter Parameter zur Diagnose von Diabetes mellitus (HbA1c-Wert). Diese nicht-enzymatische, posttranslationale Modifizierung von Proteinen wird allgemein als Glykierung bezeichnet und kann die Funktionalität von Proteinen beeinträchtigen. Deshalb wird untersucht, ob die Trübung der Augenlinsen, die Versteifung von Blutgefäßen oder Schädigungen von Nervenzellen durch eine erhöhte Glykierung verursacht werden. Diese Veränderungen treten im Alter und bei Stoffwechselkrankheiten wie Diabetes mellitus und Urämie auf, die durch eine erhöhte Glucosekonzentration bzw. die Anreicherung von 1,2-Dicarbonylverbindungen im Blut gekennzeichnet sind. Zwar gibt es Publikationen zum Vorkommen N-terminaler Amadori-Produkte an Hämoglobin und in Lebensmitteln, aber die Bildung N-terminaler AGEs wurde bisher nur in wenigen Studien untersucht. Deshalb waren die Bildung und das Vorkommen N-terminaler AGEs im physiologischen Modell, in Hämoglobin und in Backwaren Gegenstand der vorliegenden Arbeit. In der vorliegenden Arbeit wurde erstmals systematisch die Sequenzabhängigkeit der Bildung der Fructosylderivate bzw. der CM-Derivate in Konkurrenz zu den Glyoxal-2(1H)-Pyrazinonen am N-Terminus von Peptiden unter physiologischen und backtechnologischen Bedingungen untersucht. Dabei wurde nachgewiesen, dass die Variation der C-terminalen Aminosäure in Dipeptiden den Glykierungsgrad und das Produktspektrum erheblich beeinflusst. Mit dem konsequenten Nachweis der N-terminalen von Glyoxal und Methylglyoxal ableitbaren Carboxyalkylderivate und 2(1H)-Pyrazinone in humanen Hämoglobin wurde die Relevanz der N-terminalen Glykierung in vivo untermauert. Damit wird eine umfassendere Beurteilung des Dicarbonylstresses und der Glykierung insbesondere bei Urämikern und Diabetikern ermöglicht. Am Beispiel von Backwaren wurde für Lebensmittel gezeigt, dass unter trockenen Reaktionsbedingungen die 2(1H)-Pyrazinone und in wasserhaltigen Systemen die Carboxyalkylderivate bevorzugt zu erwarten sind.

AGEs accumulate in the Bruch's membrane with a detrimental effect on RPE function with age. Receptor for AGEs (RAGE) is hypothesised to have an important role in RPE dysfunction and AMD pathogenesis. It is reported to be highly expressed in the RPE and its activation leads to the induction of pro-inflammatory cytokines and oxidative stress in many other tissues. SI OOB is a ligand for RAGE and its role in retinal inflammation is not clear. This project investigated the link between RAGE activation by S100B and how this relates to RPE dysfunction and the pathogenesis of AMD. Serum analysis using ELISA showed that S100B was significantly elevated in nvAMD with no significant changes in sRAGE levels. Laser induced CNV in RAGE -/- and WT mice showed that the genetic depletion of RAGE results in smaller lesion size and concomitant infiltration of macrophages into the sub-retinal space. RAGE knock-down in endothelial cells (HMEC-l) was achieved by siRNA and SI OOB treatment almost abolished VEGF secretion and angiogenesis in RAGE knockdown cells compared to control cells which was measured by migration and tube formation. This demonstrates that RAGE is essential for SI OOB induced signal transduction and angiogenic activity. Signalling studies showed that MAPK and AKT were less phosphorylated in RAGE knocked-down cells compared to controls with SI OOB treatment resulting in less NFK,B activation and pro-inflammatory cytokines. Caspase-3 was activated after prolonged exposure to SI OOB indicating that SI 00-RAGE mediates RPE apoptosis. This data was further supported by microarray analysis of the same group of cells. Overall, this thesis supports the hypothesis that RAGE plays an important role in RPE dysfunction and several inflammation-mediated aspects of AMD. At least in part, RAGE activation is mediated through SI OOB . This axis could play a hitherto unrecognised role in RPE age-related dysfunction and, importantly, the pathogenesis of AMD.

Traumatic brain injury (TBI) has become an increasingly unmet clinical need due to intense military conflicts worldwide. Directly impacted brain cells suffer massive death, with neighboring cells succumbing to progressive neurodegeneration accompanied by inflammatory and other secondary cell death events. Subsequent neurodegenerative events may extend to normal areas beyond the core of injury, thereby exacerbating the central nervous system’s inflammatory response to TBI. Recently CD-36 (cluster of differentiation 36/fatty acid translocase (FAT), a class B scavenger receptor of modified low-density lipoproteins (mLDLs) in macrophages, has been implicated in lipid metabolism, atherosclerosis, oxidative stress, and tissue injury in cerebral ischemia, and in certain neurodegenerative diseases. Accordingly, we proposed that CD-36 has a pivotal role in the neuroinflammatory cascade that further contributes to the pathology of TBI. First, we explored the neuroinflammatory role of CD-36 after acute and chronic stages of TBI. Second, we employed a neuroinflammatory model to test the therapeutic effect of the soluble receptor of advanced end-glycation product (sRAGE); previously shown to abrogate increased CD-36 expression in stroke. Third, we further examined ameliorating TBI related inflammation as a therapeutic pathway by combination of stem cell therapy and sRAGE. At acute stages of TBI, we observed brain co-localization of CD-36, monocyte chemoattractant protein 1 (MCP-1) and ionized calcium-binding adapter molecule 1 (Iba-1) on impacted cortical areas, significant increases of CD-36 and MCP-1 positive cells in the ipsilateral vs. contralateral hemispheres of TBI animals in acute, but no significant increases of Iba-1 expressing cells over time. In early acute stages of TBI immunoblotting showed overexpression of CD-36 in brain cortex when comparing ipsilateral and contralateral hemispheres vs. sham. Spleen CD-36 protein expression at acute post-TBI stages showed no significant difference between TBI and sham groups. In addition, immunohistochemistry revealed minimal CD-36 detection on the cortical area of impact on our chronic group. Spleen immunohistochemistry also showed co-localization of CD-36 and MCP-1 in the red pulp of spleen in acute stages of TBI animals when compared to sham. Ongoing ischemic and hyperlipidemic rodent models suggest that infiltrating monocytes/macrophages from the periphery are the major source of CD-36 in the post-ischemic brain. Likewise, CD-36 expressing monocytes in the spleen after TBI may suggest its role in peripheral immune response, which may exacerbates the inflammatory response after TBI. Therefore, CD-36 may play a key role as a pathological link between inflammation and TBI. Our results suggest an intimate involvement of CD-36 mediated inflammation in TBI, providing novel insights into the understanding of disease neuroinflammation and as a potent therapeutic target for TBI treatment. The critical timing (i.e., 24-48 hours) of CD-36 expression (from downregulation to upregulation) may signal the transition of functional effects of this immune response from pro-survival to cell death. This observed dynamic CD-36 expression also suggests the therapeutic window for TBI. The detection of CD-36 expression in brain areas proximal, as well as distal, to the site of impacted injury suggests its role in both acute and progressive evolution of TBI. CD-36 neuroinflammatory role has clinical relevance for treating patients who have suffered any TBI condition at acute and chronic stages.

Cardiovascular diseases (CVDs) are the leading cause of mortality and morbidity in the world. The risk of CVDs increases with age, tobacco, diabetes, dyslipidemia, obesity and kidney dysfunction. The incidence and prevalence of CVDs demands the development of efficient strategies for prevention and treatment, as well as new biomarkers. The receptor for advanced glycation end-products (RAGE) is implicated in several metabolic and inflammatory disorders. RAGE activation by its multiple ligands, i.e. advanced glycation end-products (AGEs), S100 proteins and amphoterin (HMGB1) induces pro-inflammatory events upon RAGE engagement. The soluble circulating form of RAGE (sRAGE) has been proposed as a biomarker of vascular risk, disease severity and outcome, especially in individuals with diabetes or kidney dysfunction. However, data is controversial since positive and negative correlations are observed for a same disease. Nevertheless, the importance of the ligand-RAGE axis in pathological processes and the wide range of RAGE-binding molecules (from pro-inflammatory proteins to autoantibodies), appreciates the present study.In this thesis, we first investigated effects of RAGE ligands and the recently described anti-sRAGE autoantibodies on sRAGE quantification. We hypothesized that interactions between sRAGE and these molecules could impair sRAGE quantification. On the second part, we evaluated the value of sRAGE and anti-sRAGE autoantibodies as biomarkers of metabolic improvement after bariatric surgery for morbid obesity. Patients were selected from the established cohort ABOS (Lille). RAGE ligands (Nε-carboxymethyllysine, S100A6, S100A12, S100B, HMGB1 and amyloid beta peptide) bind sRAGE at different sites and could potentially impair its quantification through epitope masking. We tested this hypothesis by incubating these ligands, from physiological to pathological concentrations, with recombinant sRAGE and serum to evaluate their effects on sRAGE quantification. Anti-sRAGE autoantibodies were identified and further purified and their effects on sRAGE measurement evaluated. The presence of ligands or anti-sRAGE autoantibodies did not impair recombinant or serum sRAGE quantification. Obesity is a condition of dyslipidemia, glycemia deregulation and inflammation where RAGE is believed to play an important role. We aimed then to investigate the levels of sRAGE and its autoantibodies according to metabolic improvement in obese subjects submitted to weight loss surgery. Patients were highly selected from a well established cohort (morbidly obese patients eligible for gastric bypass, ABOS, Lille). Patients under statins treatment, with kidney dysfunction or hypertension, factors that could affect sRAGE levels, were excluded. In obese patients, significant higher levels of sRAGE and anti-sRAGE autoantibodies were observed before weight-loss surgery. In parallel to body-mass Index, both sRAGE and anti-sRAGE titers were significantly decreased one year after surgery.We demonstrate that the variations of sRAGE levels among the literature are, most likely, not due to an interaction between RAGE ligands and sRAGE. Other hypothesis like the regulation of sRAGE formation and clearance are further discussed. We have, for the first time demonstrated the presence of anti-sRAGE autoantibodies in obese subjects and that their levels decrease after bariatric surgery. Although our data suggest that morbid obese status leads to an autoimmune reactions against sRAGE. Together, our findings argue against sRAGE as a good biomarker but suggest that anti-sRAGE autoantibodies may have a potential implication to evaluate metabolic risk and autoimmunity associated to RAGE

Produtos de glicação avançada (AGE) prejudicam o transporte reverso de colesterol, por diminuírem o efluxo de colesterol de macrófagos, mediado por HDL. Neste estudo, avaliamos o papel da albumina modificada por glicação avançada (albumina-AGE) sobre a geração de espécies reativas de oxigênio (ROS) pela mitocôndria e pelo sistema NADPH oxidase, e sua implicação sobre o conteúdo do receptor de HDL (ABCA-1) em macrófagos. Albumina-AGE foi preparada pela incubação com glioxal (GO), metilglioxal (MGO) ou glicolaldeído (GAD) e albumina controle (albumina C), com tampão fosfato apenas. Albumina C e AGE foram incubadas com macrófagos, ao longo do tempo, para determinação da produção de ROS e do conteúdo de ABCA-1, por citometria de fluxo. Macrófagos tratados com albumina-GO, MGO e GAD apresentaram aumento na produção de ROS de, respectivamente, 24%, 25% e 24%, em comparação às células tratadas com albumina C. A elevação na produção de ROS foi prevenida pelo tratamento celular com inibidor de NADPH oxidase ou desacoplador mitocondrial, evidenciando o papel da NADPH oxidase e da mitocôndria na geração de ROS induzida pela albumina-AGE. Em comparação com as células tratadas com albumina-C, a respiração mitocondrial basal, determinada por oxigrafia, foi reduzida em 35% e 46% nas células expostas, respectivamente, à albumina-GO e albumina-GAD e não foi restabelecida após tratamento celular com desacoplador mitocondrial. O conteúdo total de proteínas carboniladas aumentou 41% em macrófagos tratados com albumina-GAD, em comparação à albumina-C. A redução no conteúdo de ABCA-1, observada após 8 horas de tratamento com albumina-GAD, foi paralela ao incremento na produção de ROS, sendo prevenida pelo tratamento com aminoguanidina, a qual também diminuiu a geração de ROS em macrófagos tratados com albumina-AGE. Por outro lado, a benfotiamina não conseguiu restaurar o conteúdo de ABCA-1, o que foi associado à menor redução na geração de ROS, promovida por este fármaco. Os resultados apontam para papel da albumina-AGE na diminuição do efluxo de colesterol celular, notadamente por reduzir a expressão de ABCA-1, vinculada ao aumento do estresse oxidativo em macrófagos. A inibição do estresse oxidativo, induzido pela albumina-AGE, previne distúrbios no transporte reverso de colesterol por impedir a redução de ABCA-1, contribuindo assim para prevenir a aterosclerose no diabete melito
Advanced glycation end products (AGE) impair reverse cholesterol transport, by decreasing the HDL-mediated cholesterol efflux from macrophages. We evaluated the role of advanced glycated albumin (AGE-albumin) on the generation of reactive oxygen species (ROS) by mitochondria and NADPH oxidase, and its implication on the HDL receptor (ABCA-1) level in macrophages. AGE-albumin was prepared by incubation with glyoxal (GO), methylglyoxal (MGO) or glycolaldehyde (GAD) and control albumin (C-albumin) with phosphate buffered saline alone. C and AGE-albumin were incubated along time with J774 macrophages in order to determine ROS production and ABCA-1 protein level by flow citometry. Macrophages treated with GO, MGO and GAD-albumin presented, respectively, 24%, 25% and 24% increased ROS production compared to cells treated with C-albumin. The increase in ROS production was prevented by cell treatment with a NADPH oxidase inhibitor or mitochondrial uncoupler, demonstrating a role of NADPH oxidase and mitochondria in AGE-albumin-induced ROS generation. Compared to cells treated with C-albumin, basal mitochondrial respiration, determined by oxygraphy, was 35% and 46% reduced in cells exposed, respectively, to GO and GAD-albumin and was not restored after cell treatment with mitochondrial uncoupling. Intracellular carbonyl content increased 41% in macrophages treated with GAD-albumin as compared to C-albumin. In macrophages treated with GAD-albumin, the reduction in ABCA-1 content observed after 8 hours of treatment was accompained by the increase of ROS production. Aminoguanidine that prevented ROS generation was able to restore ABCA-1 levels. On the other hand, benfotiamine failed to restore ABCA-1 protein levels which was ascribed to a lesser reduction in ROS generation by this compund. These results point to a role of AGE-albumin on the reduction of cellular cholesterol efflux, notably by diminishing ABCA-1 protein level in macrophages which is associated with intracellular oxidative stress. Inhibition of oxidative stress induced by AGE-albumin prevents disturbances in reverse cholesterol transport by curbing the reduction of ABCA-1 elicited by advanced glycation in macrophages and therefore may contribute to the prevention of atherosclerosis in diabetes mellitus

Furosin entsteht bei der Salzsäurehydrolyse aus den Amadori-Produkten des Lysins und wird als Marker für den Fortschritt der frühen Maillard-Reaktion, zur Beurteilung von lebensmitteltechnologischen Prozessen sowie zur Berechnung des verfügbaren und des nicht verfügbaren Lysins in Lebensmitteln verwendet. Für die Nutzung von Furosin als Qualitätsparameter ist die reproduzierbare und konstante Bildung während der Salzsäurehydrolyse entscheidend. Dies wird in der Literatur jedoch kontrovers diskutiert. Im ersten Abschnitt dieser Arbeit galt es deshalb, die molaren Ausbeuten an Furosin und den weiteren Hydrolyseprodukten Lysin, Pyridosin und N[epsilon]-Carboxymethyl-lysin zu bestimmen und damit eine sichere Interpretation der Ergebnisse zu ermöglichen. Dazu wurden peptid-gebundene Amadori-Produkte des N[alpha]-Hippuryl-lysins in chromatographisch reiner Form dargestellt. Weiterhin wurden N[alpha]-Hippuryl-N[epsilon]-carboxymethyl-lysin und Pyridosin als Standard gewonnen. Bei den Hydrolyseexperimenten zeigten die Fructosyl-Amadori-Produkte ein ähnliches Verhalten. Nach Hydrolyse mit 6M Salzsäure wurden molare Ausbeuten an Furosin von 32% für Fructosyl-lysin und jeweils 34% für Lactulosyl- und Maltulosyl-lysin bestimmt. Signifikant höhere Ausbeuten an Furosin waren nach Hydrolyse mit 8M Salzsäure festzustellen, 46% für Fructosyl-lysin, 50% für Lactulosyl-lysin und 51% für Maltulosyl-lysin. Im Gegensatz zu den Fructosyl-Derivaten war die molare Ausbeute an Furosin bei Tagatosyl-lysin unabhängig von der verwendeten Salzsäurekonzentration (6 bis 8M) und wurde zu 42% bestimmt. Anhand der auf Basis der molaren Ausbeuten ermittelten Überführungsfaktoren kann nun erstmals die Lysin-Derivatisierung mittels der Analytik von Furosin sicher bestimmt werden. Das ermöglicht exakte Aussagen zum Fortschritt nichtenzymatischer Glykierungsreaktionen sowohl in Lebensmittel als auch in vivo. Aufgrund der Relevanz für biologische Systeme und für Lebensmittel wurden weiterhin Reaktionen von alpha-Dicarbonylverbindungen mit kurzkettigen Peptiden und dem Protein Insulin unter physiologischen Bedingungen (pH=7,4 und 37°C) untersucht. Bei der Reaktion von Glyoxal mit ausgewählten Tripeptiden wurde eine sehr schnelle Derivatisierung der Peptide und jeweils die gleichzeitige Bildung eines definierten Produktes festgestellt. Mittels nuklearmagnetischer Resonanzspektroskopie und massenspektroskopischer Analyse konnten die Produkte zweifelsfrei, jeweils als die am N-Terminus durch einen 2(1H)-Pyrazinon-Ring modifizierten Peptide, aufgeklärt werden. Das Hauptprodukt der Reaktion von Methylglyoxal mit dem Peptid Gly-Ala-Phe wurde ebenfalls als 2(1H)-Pyrazinon-Peptid aufgeklärt. Nach Inkubation von Insulin mit Glyoxal unter physiologischen Bedingungen in verdünnter Lösung konnte weiterhin gezeigt werden, dass die 2(1H)-Pyrazinon-Bildung ebenfalls an einem Protein erfolgt. Die identifizierten N-terminalen 2(1H)-Pyrazinone weisen charakteristische UV-Absorptions- sowie Fluoreszenz-Spektren auf. Um die Reaktivität des N-Terminus und damit die Bedeutung der 2(1H)-Pyrazinon-Bildung beurteilen zu können, wurden vergleichende Studien mit dem als Hauptreaktionspartner für alpha-Dicarbonylverbindungen angesehenen Arginin durchgeführt. Bei diesen Experimenten zeigte der N-Terminus und peptidgebundenes Arginin eine nahezu identische Reaktivität. Auf Grund dieser Ergebnisse ist fest davon auszugehen, dass es sich bei den identifizierten N-terminalen 2(1H)-Pyrazinonen um eine neue Klasse von sogenannten Advanced Glycation Endproducts (AGEs) mit Bedeutung in physiologischen Systemen und in Lebensmitteln handelt.

Furosin entsteht bei der Salzsäurehydrolyse aus den Amadori-Produkten des Lysins und wird als Marker für den Fortschritt der frühen Maillard-Reaktion, zur Beurteilung von lebensmitteltechnologischen Prozessen sowie zur Berechnung des verfügbaren und des nicht verfügbaren Lysins in Lebensmitteln verwendet. Für die Nutzung von Furosin als Qualitätsparameter ist die reproduzierbare und konstante Bildung während der Salzsäurehydrolyse entscheidend. Dies wird in der Literatur jedoch kontrovers diskutiert. Im ersten Abschnitt dieser Arbeit galt es deshalb, die molaren Ausbeuten an Furosin und den weiteren Hydrolyseprodukten Lysin, Pyridosin und N[epsilon]-Carboxymethyl-lysin zu bestimmen und damit eine sichere Interpretation der Ergebnisse zu ermöglichen. Dazu wurden peptid-gebundene Amadori-Produkte des N[alpha]-Hippuryl-lysins in chromatographisch reiner Form dargestellt. Weiterhin wurden N[alpha]-Hippuryl-N[epsilon]-carboxymethyl-lysin und Pyridosin als Standard gewonnen. Bei den Hydrolyseexperimenten zeigten die Fructosyl-Amadori-Produkte ein ähnliches Verhalten. Nach Hydrolyse mit 6M Salzsäure wurden molare Ausbeuten an Furosin von 32% für Fructosyl-lysin und jeweils 34% für Lactulosyl- und Maltulosyl-lysin bestimmt. Signifikant höhere Ausbeuten an Furosin waren nach Hydrolyse mit 8M Salzsäure festzustellen, 46% für Fructosyl-lysin, 50% für Lactulosyl-lysin und 51% für Maltulosyl-lysin. Im Gegensatz zu den Fructosyl-Derivaten war die molare Ausbeute an Furosin bei Tagatosyl-lysin unabhängig von der verwendeten Salzsäurekonzentration (6 bis 8M) und wurde zu 42% bestimmt. Anhand der auf Basis der molaren Ausbeuten ermittelten Überführungsfaktoren kann nun erstmals die Lysin-Derivatisierung mittels der Analytik von Furosin sicher bestimmt werden. Das ermöglicht exakte Aussagen zum Fortschritt nichtenzymatischer Glykierungsreaktionen sowohl in Lebensmittel als auch in vivo. Aufgrund der Relevanz für biologische Systeme und für Lebensmittel wurden weiterhin Reaktionen von alpha-Dicarbonylverbindungen mit kurzkettigen Peptiden und dem Protein Insulin unter physiologischen Bedingungen (pH=7,4 und 37°C) untersucht. Bei der Reaktion von Glyoxal mit ausgewählten Tripeptiden wurde eine sehr schnelle Derivatisierung der Peptide und jeweils die gleichzeitige Bildung eines definierten Produktes festgestellt. Mittels nuklearmagnetischer Resonanzspektroskopie und massenspektroskopischer Analyse konnten die Produkte zweifelsfrei, jeweils als die am N-Terminus durch einen 2(1H)-Pyrazinon-Ring modifizierten Peptide, aufgeklärt werden. Das Hauptprodukt der Reaktion von Methylglyoxal mit dem Peptid Gly-Ala-Phe wurde ebenfalls als 2(1H)-Pyrazinon-Peptid aufgeklärt. Nach Inkubation von Insulin mit Glyoxal unter physiologischen Bedingungen in verdünnter Lösung konnte weiterhin gezeigt werden, dass die 2(1H)-Pyrazinon-Bildung ebenfalls an einem Protein erfolgt. Die identifizierten N-terminalen 2(1H)-Pyrazinone weisen charakteristische UV-Absorptions- sowie Fluoreszenz-Spektren auf. Um die Reaktivität des N-Terminus und damit die Bedeutung der 2(1H)-Pyrazinon-Bildung beurteilen zu können, wurden vergleichende Studien mit dem als Hauptreaktionspartner für alpha-Dicarbonylverbindungen angesehenen Arginin durchgeführt. Bei diesen Experimenten zeigte der N-Terminus und peptidgebundenes Arginin eine nahezu identische Reaktivität. Auf Grund dieser Ergebnisse ist fest davon auszugehen, dass es sich bei den identifizierten N-terminalen 2(1H)-Pyrazinonen um eine neue Klasse von sogenannten Advanced Glycation Endproducts (AGEs) mit Bedeutung in physiologischen Systemen und in Lebensmitteln handelt.

Asthma is a heterogeneous disorder encompassing distinct clinical phenotypes thought to be mediated by distinct mechanisms. The Receptor for Advanced Glycation End products (RAGE) is a pattern-recognition receptor capable of ‘sensing’ exogenous and endogenous molecules; and there is evidence that ligand-RAGE axis is activated in asthma. We investigated the role of RAGE and its ligand high-mobility group box-1 (HMGB1) in the inception and progression of allergen and virus-induced asthma using mouse models. We demonstrate that RAGE is a critical mediator of allergic airway sensitization induced by house dust mite (HDM) and cockroach (CR) allergens. Our studies suggest that RAGE is engaged secondary to the release of HMGB1 by airway epithelial cells; and that HMGB1-RAGE signalling drives type 2 immunity and airway inflammation. RAGE is also required for anti-viral immunity to pneumonia virus of mice (PVM) (equivalent to human respiratory syncytial virus). PVM infection in RAGE-deficient mice led to increased HMGB1 expression in the airways; this contributed to the development of an asthma-like pathology characterised by airway smooth muscle remodelling, airways hyperresponsiveness and the absence of granulocytic inflammation, representing a pauci-granulocytic phenotype of human asthma. In separate studies we investigated whether an anti-IL-6 receptor (IL-6R) antibody protects against allergic airway inflammation. Intriguingly, anti-IL-6R protected against CR-induced airway inflammation but exacerbated the airway inflammatory response to HDM. This differential response was related to differential activation of IL-6 signalling mechanisms in response to CR and HDM. The studies herein expose novel molecular mechanisms that may explain the development of specific phenotypes of human asthma in response to viral or allergic triggers. Selective targeting of these pathways in appropriate patient sub-groups may lead to better outcomes in asthma management.

Le syndrome de détresse respiratoire aiguë (SDRA) est caractérisé par des lésions alvéolaires diffuses menant à un œdème alvéolaire lésionnel et une insuffisance respiratoire aiguë hypoxémique. Malgré les progrès récents dans la prise en charge des patients de réanimation, le SDRA reste un syndrome fréquent et associé à une morbimortalité importante. Deux mécanismes principaux du SDRA semblent associés à une mortalité plus élevée et à des réponses thérapeutiques différentes : la déficience de la clairance liquidienne alvéolaire (AFC, pour alveolar fluid clearance), l’incapacité pour l’épithélium alvéolaire de résorber l’œdème alvéolaire, et la présence d’un phénotype « hyper-inflammatoire ». Les approches pharmacologiques du traitement du SDRA restent limitées et il est nécessaire de poursuivre l’étude des voies biologiques impliquées dans la pathogénie du SDRA et dans sa résolution afin de développer des approches innovantes des prises en charge diagnostique et thérapeutique du SDRA. RAGE, le récepteur des produits de glycation avancée, est un récepteur multi-ligands, exprimé abondamment par les cellules épithéliales alvéolaires du poumon (pneumocytes), qui module de nombreuses voies de signalisation intracellulaire. De nombreuses études récentes suggèrent que sRAGE, la forme soluble principale de RAGE, pourrait servir de marqueur lésionnel du pneumocyte de type I, et que RAGE pourrait jouer un rôle-pivot dans la pathophysiologie du SDRA, en initiant et en entretenant la réponse inflammatoire alvéolaire. Nos objectifs étaient de caractériser les rôles de RAGE au cours du SDRA, grâce à une approche translationnelle combinant études cliniques et précliniques. D’abord, des études cliniques observationnelles et interventionnelles ont été conduites afin de caractériser sRAGE comme un véritable biomarqueur dans le SDRA. Ensuite, des cultures in vitro de cellules épithéliales et de macrophages, ainsi qu’un modèle expérimental in vivo de SDRA murin par instillation trachéale d’acide chlorhydrique ont été utilisés pour décrire les effets de la voie RAGE sur les mécanismes d’AFC et l’inflammation macrophagique médiée par l’inflammasome « Nod-Like Receptor family, Pyrin domain containing 3 » (NLRP3). Enfin, l’effet d’une inhibition de RAGE, par sRAGE recombinant ou par anticorps monoclonal anti-RAGE, était testée en modèle murin. Nos résultats issus des études cliniques suggèrent que sRAGE présente toutes les caractéristiques d’un biomarqueur au cours du SDRA, avec un intérêt dans le diagnostic, le pronostic et la prédiction du risque de développer un SDRA dans une population à risque. Pris ensemble, notre travail suggère que la voie RAGE joue un rôle important dans la régulation de l’atteinte pulmonaire, de l’AFC et de l’activation macrophagique au cours du SDRA. Toutefois, les mécanismes précis de cette régulation restent incertains. La forme soluble de RAGE (sRAGE), lorsqu’elle est dosée dans le plasma, présente toutes les caractéristiques d’un biomarqueur pouvant être utile en pratique clinique, mais son intérêt dans la sélection de sous-groupes (ou « phénotypes ») de patients pouvant bénéficier de traitements ciblés reste à étudier. La voie RAGE pourrait enfin représenter une cible thérapeutique prometteuse. Bien que des études de validation restent nécessaires, ces résultats pourraient ouvrir de nouvelles perspectives dans la prise en charge des patients atteints de SDRA
The acute respiratory distress syndrome (ARDS) is associated with diffuse alveolarinjury leading to increased permeability pulmonary edema and hypoxemic respiratory failure. Despite recent improvements in intensive care, ARDS is still frequent and associated with high mortality and morbidity. Two major features of ARDS may contribute to mortality and response to treatment: impaired alveolar fluid clearance (AFC), i.e. altered capacity of the alveolar epithelium to remove edema fluid from distal lung airspaces, and phenotypes of severe inflammation. Pharmacological approaches of ARDS treatment are limited and further mechanistic explorations are needed to develop innovative diagnostic and therapeutic approaches. The receptor for advanced glycation endproducts (RAGE) is a multiligand pattern recognition receptor that is abundantly expressed by lung alveolar epithelial cells andmodulates several cellular signaling pathways. There is growing evidence supporting sRAGE (the main soluble isoform of RAGE) as a marker of epithelial cell injury, and RAGE may be pivotal in ARDS pathophysiology through the initiation and perpetuation of inflammatory responses. Our objectives were to characterize the roles of RAGE in ARDS through a translational approach combining preclinical and clinical studies. First, observational and interventional clinical studies were conducted to test sRAGE as a biomarker during ARDS.Then, cultures of epithelial cells, macrophages and a mouse model of acidinduced lung injury were used to describe the effects of RAGE pathway on AFC and inflammation, with special emphasis on a macrophage activation through NodLikeReceptor family, Pyrindomain containing 3 (NLRP3) inflammasome. Acidinjured mice were treated with an antiRAGE monoclonal antibody or recombinant sRAGE to test the impact of RAGE inhibition on criteria of experimental ARDS. Results from clinical studies support a role of sRAGE as a biomarker of ARDS, withdiagnostic, prognostic and predictive values. In addition, plasma sRAGE is correlated with a lung imaging phenotype of nonfocal ARDS and could inform on therapeutic response. Herein, we also describe in vivo and in vitro effects of RAGE activation on transepithelial fluid transport and expression levels of epithelial channels (aquaporin 5, αNa,KATPaseandαENaC) and on macrophage activation through NLRP3 inflammasome. Finally, RAGE inhibition improves AFC and decreases lung injury in vivo. Taken together, our findings support a role of RAGE pathway in the regulation of lung injury, AFC and macrophage activation during ARDS, albeit precise regulatory mechanisms remain uncertain. sRAGE has most features of a validated biomarker that could be used in clinical medicine, but whether it may help to identify subgroups (or phenotypes) of patients that would benefit from tailored therapy remains underinvestigated. Modulation ofRAGE pathway may be a promising therapeutic target, and though validation studies are warranted, such findings may ultimately open novel diagnostic and therapeutic perspectivesin patients with ARDS

Τα τελικά προϊόντα προχωρημένης γλυκοζυλίωσης (AGEs: Advanced Glycation Endproducts) παίζουν σημαντικό ρόλο στην παθογένεια των διαβητικών αγγειακών επιπλοκών. Το καλύτερα χαρακτηριζόμενο είναι η N-καρβοξυμεθυλ-λυσίνη (CML). Τα AGEs προκαλούν σημαντικές επιδράσεις στα αγγεία με την πρόσδεσή τους σε ειδικούς υποδοχείς της κυτταρικής επιφάνειας, όπως τον RAGE (Receptor for Advanced Glycation Endproducts). Διαλυτές μορφές του RAGE (sRAGE) εμφανίζονται στο ανθρώπινο αίμα και δρουν ως παγίδα αιχμαλωτίζοντας τους φλεγμονώδεις προσδέτες του RAGE εξωκυττάρια, προστατεύοντας με αυτό τον τρόπο τα κύτταρα από τη βλάβη που προάγεται από τα AGEs. Σκοπός αυτής της εργασίας ήταν να μελετηθούν τα επίπεδα του sRAGE, η πρωτεϊνική έκφραση του RAGE, καθώς και τα επίπεδα CML σε σχέση με διάφορες κλινικές και βιοχημικές παραμέτρους σε παιδιά, εφήβους και νεαρούς ενήλικες με ΣΔ1. Τα επίπεδα sRAGE και CML προσδιορίστηκαν με ELISA και η πρωτεϊνική έκφραση του RAGE στα μονοπύρηνα του περιφερικού αίματος με ανοσοαποτύπωση κατά Western σε 74 παιδιά, εφήβους και νεαρούς ενήλικες με ΣΔ1 (13± 4 χρονών) και 43 μάρτυρες αντίστοιχης ηλικίας, φύλου και σταδίου Tanner. Σ’ αυτή την εργασία τα αυξημένα επίπεδα sRAGE στα παιδιά με ΣΔ1 και πιο ειδικά, σ’ αυτά ηλικίας κάτω από 13 ετών και με διάρκεια διαβήτη κάτω από 5 έτη, μπορεί να είναι ένα προσωρινό προστατευτικό μέτρο ενάντια στην κυτταρική βλάβη και πιθανόν να είναι επαρκές για να εξουδετερώσει επαρκώς τα κυκλοφορούντα CML, εμποδίζοντας έτσι τις διαβητικές αγγειακές επιπλοκές. Επίσης, μια ήπια αύξηση της LDL θα μπορούσε να είναι ένα ερέθισμα για την αύξηση του sRAGE, οδηγώντας στη δέσμευση του CML και τελικά τη μείωση των επιπέδων CML στην κυκλοφορία. Τα μειωμένα επίπεδα της πρωτεϊνικής έκφρασης του RAGE 55 kd (υποδοχέα πλήρους μήκους) μπορεί να αντανακλούν την αυξημένη έκφραση του sRAGE στους ασθενείς με ΣΔ1 συνολικά λόγω της αποκοπής του RAGE με μεταλλοπρωτεϊνάσες. Με την παρουσία κάποιου παράγοντα κινδύνου, όπως αύξηση ηλικίας, περιμέτρου κοιλίας, BMI, συστολικής ή διαστολικής αρτηριακής πίεσης ή επιδείνωση λιπιδαιμικού προφίλ αυξάνεται η πρωτεϊνική έκφραση της ισομορφής αυτής, ενώ φαίνεται αντίστοιχα να μειώνονται τα επίπεδα του sRAGE. Φαίνεται τελικά ότι συνολικά στα παιδιά, τους εφήβους και τους νεαρούς ενήλικες με ΣΔ1 υπάρχει μια υποκλινική διαταραχή του άξονα sRAGE-RAGE-CML, η οποία δύναται να μετατραπεί σε κλινικά εμφανείς αγγειακές βλάβες, αν προστεθούν περαιτέρω επιβαρυντικοί παράγοντες.
The binding of Advanced Glycation Endproducts (AGEs) to their receptor (RAGE) plays a major role in the development of diabetic vascular complications. This work is based on the relation between circulating soluble RAGE (sRAGE) levels in children, adolescents and young adults with IDDM and RAGE protein expression in association with N-(carboxymethyl)lysine (CML), a major antigenic AGEs component. Circulating sRAGE and CML levels were determined by ELISA and RAGE protein expression was evaluated in peripheral blood mononuclear cells by western immunoblotting in 74 children, adolescents and young adults with IDDM (134 years old) and 43 age, sex and Tanner stage-matched controls. Serum sRAGE levels were significantly higher in IDDM than in controls, inversely correlated to diabetes duration and directly correlated to LDL levels. Furthermore, circulating CML levels were not significantly different between IDDM and controls. Also, the protein expression of the RAGE isoforms 55 kd (full-length), 64 kd and 100 kd, measured by western immunoblotting, was significantly lower in IDDM than in controls, whereas RAGE 37 kd levels were not significantly different between IDDM and controls. Finally, when there was a risk factor, such as increased age, poor lipid profile, increased BMI or waist circumference or increased systolic or diastolic pressure, then it seemed that isoforms RAGE 55, 64 and 100 kd were increased. Isoform RAGE 64 kd could be RAGE-v5, a splice variant which resulted in a change of amino acid sequence in the extracellular ligand-binding domain of RAGE. Isoform RAGE 37 kd seemed to be Δ8-RAGE, a soluble splice variant with probably protective function, which had been found increased in patients with increased HDL. Finally, isoform RAGE 100 kd seemed to be some other splice variant in peripheral mononuclear cells. In conclusion, increased serum levels of sRAGE seen in IDDM children may be a temporary protective measure against cell damage and may be sufficient to efficiently eliminate excessive circulating CML. Moreover, the lower protein expression of the full-length RAGE in IDDM may also reflect the increased sRAGE expression in patients due to RAGE cleavage by metalloproteases. Consequently, in IDDM children, adolescents and young adults there may be a subclinical perturbation of the sRAGE-RAGE-CML axis, which could lead to future clinical vascular damage if additional risk factors are added over time.