Rozprawy doktorskie na temat „Receptors for advanced glycation end product (RAGE)”

Type 2 diabetes and cigarette smoke exposure are associated with an increased risk of cardiovascular complications. The role of advanced glycation end-products (AGEs) is already well-established in numerous comorbidities including cardiomyopathy. Given the role of AGEs and their receptor, RAGE, in activating inflammatory pathways, we sought to determine whether ceramides could be a mediator of RAGE-induced altered heart mitochondrial function. Using an in vitro model, we treated H9C2 cardiomyocytes with carboxy-methyl lysine-BSA, followed by mitochondrial respiration assessment. We found that mitochondrial respiration was significantly impaired in AGE-treated cells, but not when co-treated with myriocin, an inhibitor of de novo ceramide biosynthesis. Moreover, we exposed WT and RAGE KO mice to side-stream cigarette smoke and found reduced mitochondrial respiration in the left ventricle myocardium from WT mice, but the RAGE KO mice were protected from this effect. Finally, conditional over-expression of RAGE in the lungs of mice also elicited a robust increase in left ventricular ceramides. Altogether, these findings suggest a RAGE-ceramide axis as an important contributor to cardiomyopathy.

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) has increasingly been demonstrated to be an important modulator of inflammation in cases of pulmonary disease. Published reports involving tobacco smoke exposure have demonstrated increased expression of RAGE, its participation in pro-inflammatory signaling and its role in irreversible pulmonary remodeling. The current research evaluated for the first time the in vivo effects of short-term tobacco smoke exposure in RAGE null and control mice compared to identical animals exposed to room air only. Quantitative real time PCR, immunoblotting, and immunohistochemistry revealed elevated RAGE expression in controls after four weeks of exposure and an anticipated absence of RAGE expression in RAGE null mice regardless of smoke exposure. Inflammatory cell behaviors were confirmed by measuring active Ras, NF-κB, and cytokine synthesis and secretion. Furthermore, bronchoalveolar lavage fluid (BALF) was procured from RAGE null and control animals after exposure for the assessment of total protein in order to indirectly measure vascular permeability, inflammatory cells and chemoattractant molecules involved in the inflammatory response. As a general theme, inflammation induced by tobacco smoke exposure was influenced by the availability of RAGE. These data reveal captivating information suggesting a role for RAGE signaling in lungs exposed to tobacco smoke. Furthermore, research may demonstrate RAGE signaling as an important therapeutic target capable of ameliorating cell level inflammation in those coping with exposure.

Preeclampsia (PE), intrauterine growth restriction (IUGR) and gestational diabetes (GDM) increase the risk of maternal and fetal morbidity and mortality. The roles of Advanced Glycation End-products (AGEs) are already well documented concerning inflammation, hypoxia and oxidative stress. AGEs bind to its receptor, Receptor for Advanced Glycation End-products (RAGE), and activate an inflammatory pathway. This pathway alters the efficacy of invasive trophoblast cells and in the placenta and can result in placental dysfunction. We hypothesized that the placental dysfunction found in PE, IUGR, and GDM resulted from an over activation of the RAGE-mediated inflammatory pathway. Using human placental samples, we found that RAGE protein expression via western blotting was increased in PE and decreased in IUGR while GDM remained similar to that of control placentas. We then wanted to determine the efficacy of RAGE activation to alter the invasive nature of invasive cytotrophoblasts cells. We found that the addition of AGEs to SW71 cells decreases invasion through the activation of JNK and ERK cellular signaling pathways. Altogether these findings suggest that RAGE activation in trophoblast cells seems result in insufficient placental pathogenesis causing PE, however the IUGR and GDM samples we obtained did not seem to have resulted from RAGE activation. We also found that RAGE activation can alter the ability of invasive trophoblasts to invade, thus limiting the ability of the placental cells to remodel the maternal spiral arteries. We believe that further research into specific triggers of IUGR (smoking-induced) and un-treated diabetes could result in RAGE stimulated placental insufficiency.

Receptors for advanced glycation end-products (RAGE) are pattern recognition receptors of the immunoglobulin superfamily highly expressed in the lung. Likely functions include the modulation of pulmonary inflammation during disease. However, the contributions of RAGE in the developing lung in cases where secondhand smoke (SHS) exposure occurs are unknown. In order to test the hypothesis that RAGE misexpression adversely affects lung morphogenesis, we exposed gestating dams to a controlled dose of SHS during the last four critical days of in utero lung morphogenesis. We discovered that both maternal and fetal lungs respond to SHS by up-regulating RAGE. Exposed fetuses were markedly smaller compared to controls and lungs were compromised in terms of apoptotic status, collagen abundance necessary in the derivation of respiratory compartments, and the expression of MMP-9, a protease known to target extracellular matrix. Interestingly, RAGE knock out animals similarly exposed to SHS were protected, in part, from the same SHS-mediated pulmonary abnormalities. We next generated a conditional transgenic mouse that provided an opportunity to genetically augment distal lung RAGE expression in the absence of SHS exposure. Our RAGE transgenic mice (RAGE TG) were severely hypoplastic and ultrastructural analysis demonstrated weakened basement membranes in RAGE TG animals compared to controls. Specific observations in RAGE TG mice included diminished type IV collagen required for basement membrane derivation, augmented MMP-9 expression, and inhibition of pulmonary vasculature visualized by Pecam-1 staining, a marker of vascular endothelial cells. The further observation that FoxM1, a critical transcriptional regulator of endothelial cell differentiation, was inhibited in RAGE TG mice suggested a novel potential mechanism of impaired vascularization mediated by RAGE. These data provide evidence that RAGE expression must be tightly regulated during lung organogenesis. Furthermore, additional research into the nuances of RAGE signaling during development may shed needed light on the pathobiochemistry of adult lung diseases that potentially have in utero origins.

Receptors for advanced glycation end-products (RAGE) are multi-ligand cell surface receptors of the immunoglobin family expressed by epithelium and macrophages. RAGE expression increases following ligand binding and when diverse cells are exposed to a variety of insults including cigarette smoke extract (CSE). The current research sought to characterize the pro-inflammatory contributions of RAGE expressed by alveolar macrophages (AMs) following CSE exposure. Acute exposure of mice to CSE via nasal instillation revealed diminished bronchoalveolar lavage (BAL) cellularity and fewer AMs in RAGE null mice compared to controls. Primary AMs were obtained from BAL, exposed to CSE in vitro, and RNA, DNA, and protein were analyzed. CSE significantly increased RAGE expression by wild type AMs. Employing ELISAs, wild type AMs exposed to CSE had increased levels of active Ras, a small GTPase that perpetuates pro-inflammatory signaling. Conversely, RAGE null AMs had less Ras activation compared to wild type AMs after exposure to CSE. In RAGE null AMs, assessment of p38 MAPK and NF-κB, important intracellular signaling intermediates induced during an inflammatory response, revealed CSE-induced inflammation occurs at least in part via RAGE signaling. For example, activated p38 was diminished in RAGE null AMs compared to controls and assessment of phosphorylated NF-κB in CSE exposed RAGE null AMs suggest lessened nuclear translocation of NF-κB compared to wild type AMs exposed to CSE. Importantly, quantitative RT-PCR revealed that mRNA expression of pro-inflammatory cytokines including TNF-α and IL-1β were detectably decreased and analysis of secreted proteins by ELISA displayed diminished IL-1β in RAGE null AMs exposed to CSE compared to CSE-exposed wild type AMs. These results reveal that primary AMs orchestrate CSE-induced inflammation, at least in part, via RAGE-mediated mechanisms.

Cyclooxygenase (COX) is the rate limiting enzyme that catalyses the production of prostanoids, which are crucial to vascular homeostasis. Evidence suggests that endothelial dysfunction and inflammation play a role in vascular complications in aging and diabetes. Previous animal studies by our laboratory at RMIT University reported enhanced COX expression with aging in rat aortas, platelets and monocytes. Potentially, alteration in COX expression may result in an imbalanced prostanoid production favoring the synthesis of vasoconstrictors and hence increase the risk of cardiovascular events in the aging population. The regulation of altered COX expression in aging, however, is not clear. It has been suggested that histone hyperacetylation may be an important mechanism that regulates COX levels during the aging process as increased histone acetylation has been shown to occur with aging. Thus, we hypothesized that COX expression is modulated by histone hyperacetylati on. This was investigated by measuring COX expression in histone hyperacetylated cultured endothelial cells. In the case of diabetes, studies have reported that the development of diabetes and its complications is associated with persistent inflammatory activity, evident with increased inflammatory markers in the circulation. COX-mediated pathways may be involved in this inflammatory process in diabetes. Furthermore, the formation of advanced glycation end products (AGEs) is accelerated in diabetes. AGEs can bind to receptors for AGEs (RAGE), which has also been suggested to play a role in inflammation in diabetes. We hypothesized that COX- and RAGE-mediated pathways contribute to increased inflammation in diabetes and potentiate the development of diabetic vascular complications. This was investigated by measuring changes in COX-mediated pathways in both rat and human diabetic models. The current thesis reports: 1) in cultured endothelial cells, histone hyperacetylation was associated with increased COX expression; 2) an overall increase in inflammation was observed in diabetes involving COX- and RAGE-mediated pathways. This was supported by increased platelet COX-1 and monocyte COX-2 levels in Zucker rats, increased monocyte COX-2 in human Type 1 diabetes and elevated plasma TXB2 and PGE2 levels in both human Type 1 and Type 2 diabetic subjects. Up-regulation of RAGE expression was further found in platelets and monocytes in both human diabetes types. When treated with NSAIDs, plasma prostanoid levels, COX and RAGE expression were reduced significantly in both platelets and monocytes in human diabetic subjects. 3) It is unclear how COX and RAGE expression was regulated, but histone modifications may be one of the mechanisms. Data from cultured cells indicated that increased COX expression was associated with increased histone acetylation levels induced by TSA. Concurrent increases in histone acetylation and COX-2 levels were also observed in human Type 1 diabetes, but similar findings were not observed in human Type 2 diabetes. In addition, we failed to find an age-dependent increase in monocyte histone H4 acetylation in human Type 2 diabetes despite an age-dependent increase in monocyte COX-2 expression. Thus, whether histone hyperacetylation modulates COX expression and in what conditions require further investigation.

Receptors for advanced glycation end-products (RAGE) are multi-ligand cell surface receptors highly expressed in the lung that modulate pulmonary inflammation during disease. However, the contributions of RAGE signaling are unknown during pulmonary organogenesis. In order to test the hypothesis that RAGE misexpression adversely affects lung morphogenesis, conditional transgenic mice were generated that over-express RAGE in alveolar type II cells of the lung. When RAGE is over-expressed throughout embryogenesis, severe lung hypoplasia ensues, culminating in perinatal lethality. Flow cytometry and immunohistochemistry employing cell-specific markers for various distal cell types demonstrated anomalies in key epithelial cell populations resulting from RAGE up-regulation through embryonic (E) 18.5. Electron microscopy also identified significant morphological disturbances to distal cell types including separation from the basement membrane. Possible mechanisms leading to the disappearance of pulmonary tissue by increased RAGE expression were then evaluated. A time course of lung organogenesis commencing at E12.5 demonstrated that increased RAGE expression primarily alters lung morphogenesis beginning at E16.5. TUNEL immunohistochemistry and immunoblotting for active caspase-3 confirm a shift toward apoptosis in lungs from RAGE over-expressing mice when compared to wild type controls. Assaying for NF-κB also revealed elevated nuclear translocation in lungs from transgenic mice compared to controls. An RT-PCR assessment of genes regulated by NF-κB demonstrated elevated expression of Fas ligand, suggesting increased activity of the Fas-mediated signal transduction pathway in which ligand-receptor interaction triggers cell death. These data provide evidence that RAGE expression must be tightly regulated during organogenesis. Furthermore, additional elucidation of RAGE signaling potentially involved in branching morphogenesis and cell cycle abnormalities may provide insight into the progression of RAGE-mediated lung diseases.

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.

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.

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.<br>Cobre grant "P20GM109024"

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<br>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.<br>North Dakota State University. College of Health Professions<br>NIH Grant # P20 GM109024 from the National Institute of General Medicine

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.

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.

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.

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

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.

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<br>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

碩士<br>國立清華大學<br>化學系<br>104<br>The human S100 calcium-binding protein A11 (S100A11) is a member of S100 protein family. Once S100A11 proteins bind to calcium ions at EF-hand motifs, S100A11 will change its conformation promoting interaction with target proteins. The receptor for advanced glycation end products (RAGE) consists of three extracellular domains, including V domain, C1 domain and C2 domain. In this case, V domain is the target for mS100A11 binding. RAGE binds to the ligands result in cell proliferation, cell growth and several signal transduction cascades. We used NMR and fluorescence spectroscopy to demonstrate the interactions between S100A11 and V domain. The Tranilast molecule is a drug used for treating allergic disorders. We found out that V domain and Tranilast would interact with S100A11 by using 1H-15N HSQC NMR titrations. According to the results, we obtained two binary complex models from the HADDOCK program, S100A11-RAGE V domain and S100A11-Tranilast, respectively. We superimposed these two models with the same orientation of S100A11 homodimer and demonstrated that Tranilast molecule would block the binding site between S100A11 and V domain. We further utilized the WST-1 assay to indicate that Tranilast indeed can inhibit the cell proliferation which is induced by the S100A11-V domain interaction. These results will be potentially useful in the development of derivative or new anti-cancer drugs for RAGE-dependent diseases.

Dissertação de Mestrado em Bioquímica apresentada à Faculdade de Ciências e Tecnologia<br>A imunidade inata é a primeira linha de defesa efectiva de todos os organismos,englobando mecanismos moleculares e celulares com vista a uma acção imediata, reconhecendopadrões moleculares associados a patogénios de origem externa (PAMPs), bem como, padrõesmoleculares associados a uma ameaça de origem interna (DAMPs). Esse reconhecimento épossível através de diversos receptors de reconhecimento padrão (PRRs), orquestrando, assim, aactivação de respostas imunes. Um destes receptores é o receptor dos produtos de glicaçãoavançada (RAGE, também conhecido por AGER), que é um receptor, localizado à superfície dascélulas, pertencendo à superfamília de proteinas imunoglobulinas, tendo um papel central naresposta inflamatória, mediando acções na imunidade inata. Para além do mais, o receptor RAGEtem sido implicado na neurotoxicidade e activação das células da glia. É de salientar que esterecptor é expresso em muitos tipos de células incluíndo as células do sistema nervoso central(CNS) como os neurónios, microglia, astrócitos. De notar também que, sendo um receptor queinterage com multiplos ligandos, RAGE é capaz de reconhecer um largo espectro de diferentesfamílias de ligandos estruturalmente diversos, incluíndo o ligando S100β, pertencente à famíliade proteínas S100.Nos dias de hoje, o consumo inadequado e a adição de drogas de abuso, incluíndo a diçãodos estimulantes do tipo das anfetaminas (ATS), é um problema sério e verificado em todo omundo, com inegável impacto na saúde pública, direitos humanos e na segurança. A par destesaspectos, a rápida difusão e abuso de novas substâncias psicoactivas (new psychoactivesubstances, NPS), também conhecidas como “substâncias químicas de pesquisa” ou “drogasdesenhadas”, engloba a catinonas sintética, Methylenedioxypyrovalerone (MDPV), designadapor “sais de banho”, claramente parece piorar a actual situação, já que é usada como um substitutode estimulantes ilegais, como a cocaína. O acesso incrivelmente facilitado a estas catinonas, comoo MDPV, no mercado de drogas global, através das “smart shops”, internet (“darknet”), e oaspecto da “falsa legalidade” destas substâncias, assim como, o relativo baixo custo e a suaqualidade, comparativamente a outras drogas tradicionais, são factores que têm contribuído paraa sua crescente popularidade.Ultimamente, têm sido investigados novos aspectos dentro do tema das drogas de abuso.A título de exemplo, existem cada vez mais evidências no que diz respeito à methanphetamina naimunidade inata central e periférica. Contudo, os efeitos de catinonas (como o MDPV) naimunidade inata central ainda estão por investigar. Neste sentido, este trabalho tem como principalfoco, caraterizar, pela primeira vez o impacto do MDPV nas células da glia, no cortéx frontal enos intervenientes da imunidade inata, incluíndo o receptor dos productos de glicação avançada(RAGE) e o sei ligando S100 β, 24 houras após a um regime binge de MDPV.Para além disso, o perfil comportamental dos roedores após a administração do MDPVestá pouco documentado, em comparação com a METH. Assim sendo, o nosso objectivo comeste trabalho foi caraterizar, pela primeira vez, o impacto do MDPV no cortéx frontal,nomeadamente nas células da glia e nos intervenientes da imunidade inata, incluíndo o receptordos produtos de glicação avançada (RAGE) e o seu ligando S100β, nas 24 horas após um regimede “binging”. Em paralelo à administração de MDPV, também foi administrada uma dosagemigualmente aguda de METH, no sentido de aumentar os actuais conhecimentos sobre aneurotoxicidade da METH.Assim, em primeiro lugar, e no que diz respeito ao perfil comportamental, o nosso estudooferece a primeira evidência de que um episódio de administração aguda de MDPV não acarretouperturbações emocionais nem locomoras, aumentando, no entanto, e curiosamente, a actividadeexploratória nos ratinhos. Em segundo lugar, ficou demonstrado que nem o MDPV nem a METHinfluenciaram os parametros de neurotoxicidade relacionados com as células da microglia, célulasastrocíticas, fibras de mielina, bem como, com marcadores dopaminergicos. Tal foi comprovadopelos níveis inalterados de Iba-1, GFAP, S100β, MBP e TH, respectivamente. Para além do mais,concluímos que nenhuma droga influenciou marcadamente os níveis de expressão ou os níveis deproteína total correspondentes ao receptor RAGE.Finalmente, não podemos descartar a hipótese de esta janela temporal de 24 horascorresponder a uma fase prematura para observar quaisquer alterações moleculares e celularessignificativas, induzidas por ambos os psicoestimulantes, no cortéx frontal. Neste contexto,estudos posteriores serão necessários para caraterizar os efeitos, neuronais e ao nível das célulasda glia, do MDPV, que podem passar, quer pela adopção de outras janelas temporais, quer poroutros regimes de administração, ou ainda quer pela avaliação numa outra área cerebral.<br>Innate immunity is the first effective line of defense of all organisms, comprising cellularand molecular mechanisms for immediate action, recognizing exogenous pathogen associatedmolecular patterns (PAMPs), as well as, endogenous danger associated molecular patterns(DAMPs). Such detection is possible through diverse pattern recognition receptors (PRRs),thereby orchestrating the activation of innate responses. One of these receptors is the receptor foradvanced glycation end-products (RAGE, also known as AGER), which is a cell-surface receptorbelonging to the superfamily of immunoglobulin proteins, thus playing a central role in theinflammatory response mediating events of innate immunity. Moreover, RAGE has beenimplicated in the sustainment of glial activation and neurotoxicity. Noteworthy, RAGE receptorexhibits broad expression on many different cells including CNS cells, such as neurons, microgliaand astrocytes. Also, as a multiligand receptor, RAGE recognizes a broad repertoire ofstructurally different ligand families, including S100β, belonging to the S100 protein family.Nowadays, drug misuse, including amphetamine-type stimulants (ATS) addiction, is amajor worldwide issue with an undeniable impact on public health, human rights and security. Inparallel, the rapid emergence and abuse of new psychoactive substances (NPS), also termed as“research chemicals” or “design drugs”, including the synthetic cathinone,Methylenedioxypyrovalerone (MDPV), known as "bath salt", clearly seems to worsen thisscenario, as it acts as a substitute for illegal stimulant drugs such as cocaine. The incredibly easyaccess to these cathinones, including the newly synthesized MDPV, in the global drug market,through smart shops, internet (“darknet”), and the aspect of“fake legallyness” of such substances,as well as the relative affordability and better quality compared with traditional drugs, are on thebasis of some of the reasons underlying their increasing popularity.In the last decades, a great deal of attention has been drawn to the aspects underlyingmethamphetamine (METH; an ATS)-induced neurotoxicity, in many brain regions. Nevertheless,little is known regarding MDPV neurotoxicity, including in frontal cortex, which is known to beaffected by drugs of abuse. Additionally, the behavioral profile of rodents after drugadministration is poorly documented for MDPV, in comparison with METH.Additionally, novel avenues in drug addiction sciences are being explored. As anexample, there is a growing body of evidence regarding the impact of drugs of abuse includingMETH to both peripheral and central innate immunity. However, the effects of new cathinones(including MDPV) in the central innate immunity remain unknown. Therefore, this thesis aimedto characterize, for the first time, the impact of MDPV on frontal cortex glial cells and innateimmune players including receptor for advanced glycation end-products (RAGE) and its ligand S100β, within first 24 hours following a binge MDPV regimen. In addition, emotional behaviorwas assessed. A binge neurotoxic regimen of METH was also employed to deepen currentknowledge on METH neurotoxicity.Concerning the behavioral profile, we offer a first evidence that a single binge MDPVregimen did not come with any changes in both emotional and locomotor parameters, butcuriously enhanced exploratory activity in mice. Secondly, we concluded that neither drugimpose any changes in innate immunity as well as in neurotoxicity parameters includingmicroglial, astrocytic, myelin and dopaminergic markers, as seen by the unaltered levels ofRAGE, S100β, Iba-1, GFAP and, MBP and TH, respectively.One cannot exclude the hypothesis whereby this is a premature time-window to observesignificative molecular changes in frontal cortices following both stimulants. In this context,future studies are required to further characterize neuronal and glial effects of MDPV by usingother time-points, dosing regimens and brain regions.

In der vorliegenden Arbeit wurde aneurysmatisches Aortengewebe von Patienten mit bikuspider oder trikuspider Aortenklappe untersucht. Im Laufe des Lebens ist die bikuspide Aortenklappe als häufigste angeborene Anomalie des Herzens mit zahlreichen, potentiell lebensbedrohlichen Komplikationen verbunden. Betroffene Patienten zeigen eine frühere Entwicklung und rapidere Progression von Dilatationen und – im schlimmsten Fall – Dissektionen der Aorta ascendens. Die Ätiologie dessen konnte bis dato nicht ausreichend geklärt werden. Hintergrund der Studie war eine Untersuchung von Branchetti et al., wobei eine Erhöhung von RAGE im Plasma bei Patienten mit bikuspider Klappe nachgewiesen werden konnte. Daraus wurde die Hypothese entwickelt, dass eine Expressionserhöhung von RAGE und dessen Liganden AGE im Aortengewebe selbst ursächlich mit der Aortendilatation verbunden sein könnte. In Proben von 93 Patienten wurde mittels Western Blot, ELISA und Immunhistochemie die Expression von RAGE und AGE untersucht. Hierbei zeigte sich eine signifikante Expressionserhöhung beider Proteine im Aortenaneurysma bei bikuspider Klappe im Vergleich zu Patienten mit trikuspider Aortenklappe. Auch die exemplarisch angefertigten Immunhistologien stützen diese Ergebnisse. Mögliche Folgen können Steifigkeitserhöhung der Aortenwand, Aktivierung von Matrixmetalloproteinasen sowie Erhöhung des oxidativen Stresses sein. Neben der Expression im aneurysmatischen Aortengewebe wurden auch Plasmaproben hinsichtlich AGE und RAGE analysiert, wobei sich keine Erhöhung feststellen ließ. Die Ergebnisse der Studie, die eine RAGE – Erhöhung im Plasma detektierten und ihn somit als potentiellen Biomarker für eine bikuspide Klappe diskutierten, ließen sich bei der vorliegenden Untersuchung einer kleineren Stichprobe nicht bestätigen. Ebenso stellt sich die Etablierung eines Biomarkers als anspruchsvolle Aufgabe dar. Eine Eignung von RAGE als Biomarker zur Identifikation von Patienten mit bikuspider Klappe ist kritisch zu betrachten.:Inhaltsverzeichnis Bibliographische Beschreibung Abkürzungsverzeichnis 1. Einleitung 1.1. Die bikuspide Aortenklappe (BAV) 1.1.1. Prävalenz 1.1.2. Klassifikation 1.1.3. Ätiologie 1.1.4. Assoziierte Pathologien 1.1.5. Hypothesen der Dilatationsentstehung 1.1.6. Diagnostik 1.1.7. Therapie 1.2. RAGE und AGE 1.2.1. Advanced Glycation End Products (AGE) 1.2.2. Receptor for Advanced Glycation End Products (RAGE) 1.2.3. Interaktion von AGE und RAGE 1.2.4. Bezug zum thorakalen Aortenaneurysma 2. Zielstellung 3. Material 3.1. Allgemeine Geräte 3.2. Allgemeine Materialien 3.3. Allgemeine Chemikalien 3.4. Proteinextraktion 3.5. Proteinkonzentrationsbestimmung 3.6. SDS – Gelelektrophorese 3.7. Antikörper (AK) 3.8. Western Blot Analyse 3.9. Enzyme – linked Immunosorbent Assay (ELISA) 3.10. Immunhistochemie (IHC) 3.11. Software 4. Methoden 4.1. Patientenpopulation und Probengewinnung 4.2. Isolation der Proteine aus Aortengewebe 4.3. Konzentrationsbestimmung nach BCA – Methode 4.4 Elektrophoretische Auftrennung der Proteine 4.5. Detektion von AGE und RAGE mittels Western Blot Analyse 4.6. Nachweis von AGE und RAGE mittels ELISA 4.7. Immunhistochemische Färbung von AGE und RAGE 4.8. Statistische Auswertung 5. Ergebnisse 5.1. Patientenpopulation 5.2. Expression von AGE in humanen aneurysmatischen Gewebeproben der Aorta ascendens 5.2.1. Analyse der AGE – Expression mittels Western Blot 5.2.2. Analyse der AGE – Expression mittels ELISA 5.2.3. Darstellung der Lokalisation von AGE in der Aortenwand mittels Immunhistochemie 5.3. Expression von RAGE im Aortengewebe 5.3.1. Analyse der Expression von RAGE mittels Western Blot 5.3.2. Analyse der RAGE – Expression mittels ELISA 5.3.3. Darstellung der Lokalisation von RAGE in der Aortenwand mittels Immunhistochemie 5.4. Bestimmung der Plasmaspiegel von AGE und RAGE in ausgewählten Plasmaproben 6. Diskussion 6.1. Expressionserhöhung von AGE in der Aortenwand von Patienten mit BAV 6.1.1. Mögliche Ursachen der Expressionserhöhung 6.1.2. Zusammenhang von AGE und Gefäßsteifigkeit 6.2. Expressionserhöhung von RAGE im Aortengewebe von Patienten mit BAV 6.2.1. Ätiologie der Expressionserhöhung unter Einbeziehung der Liganden 6.2.2. Folgen der RAGE – Erhöhung und ihr Einfluss auf die Gefäßwand 6.3. RAGE in seiner Rolle als Biomarker Schlussfolgerung Limitationen 7. Zusammenfassung 8. Literaturverzeichnis 9. Abbildungsverzeichnis 10. Tabellenverzeichnis Erklärung über die eigenständige Abfassung der Arbeit Lebenslauf Danksagung

Adipose tissue plays an essential role in the regulation of many metabolic processes. Excess caloric intake and decreased energy expenditure cause adipocyte hypertrophy and hyperplasia, leading to inflammation of the adipose tissue, which contributes to obesity. Recent data link the Receptor for Advanced Glycation End-products (RAGE) to high fat diet (HFD)-induced obesity and subsequent metabolic dysfunction, but its function is incompletely understood. On HFD, the concentration of RAGE ligands, such as carboxy methyl lysine (CML) - advanced glycation end-products (AGE) epitopes, S100 calcium binding protein B (S100b), and high mobility group box 1 (HMGB1) increases, activating RAGE and resulting in inflammation. However, deletion of RAGE protected against the HFD-induced obesity and resulted in increased overall energy expenditure. In this study, we tested the hypothesis that the protective mechanism of RAGE deletion is due, in part, to browning which involves inducing brown adipose tissue (BAT)-like properties in white adipose tissue (WAT). The expression of uncoupling protein 1 (UCP1), which is usually only expressed in BAT, is increased in the WAT of RAGE knockout (RKO) mice. This effect was reproduced in vitro, by silencing the Ager gene in the adipocyte cell line C3H10T1/2. We propose that RAGE affects adipocyte phenotype by downregulating the expression of browning genes such as UCP1, and therefore is a key determinant of energy expenditure and adiposity. Thus, RAGE antagonism may promote BAT phenotype and function in WAT, reducing adiposity, which may have potential therapeutic implications for treating obesity.<br>2017-03-31T00:00:00Z

博士<br>國立清華大學<br>化學系<br>99<br>S100A13為一同元雙體蛋白，屬於S100家族中之EF手臂Ca2+結合蛋白。S100A13亦為S100家族中唯一參與不具訊號序列的蛋白之非典型輸出，如：fibroblast growth factors, interleukin 1α與synaptotagmins。S100A13同時也被發現於調節acidic fibroblast growth factor 與 IL-1α多元蛋白釋放複合體之形成。在形成一特殊的多元蛋白複合體後，S100A13 與 FGF1可藉由非典型輸送途徑被運送到細胞膜外，並且調控許多生物功能，如：細胞分化、糖尿病視網膜病變、血管新生、腫瘤生長、神經新生與胚胎形成。基於S100A13與FGF1在腫瘤形成扮演重要的角色，因此避免多元蛋白複合體的形成對於抑制癌症來說是一有效的策略。 Advanced glycation end products受體(receptor for advanced glycation end products, RAGE) 在許多不同疾病當中都扮演重要的角色，包括：糖尿病併發症、腫瘤外生, 慢性發炎、阿茲海默症或中樞神經系統的多發性硬化症。在與不同的配體，如S100 蛋白、醣基化蛋白、 amyloid-β與 HMGB1結合後，RAGE可誘導不同的細胞訊息傳導產生，並且在發炎反應、急性或慢性發炎病變及某些癌症中扮演中心角色。 在第二章中，我們闡明S100A13與amlexanox在結構上的交互作用。我們利用等溫滴定量熱法、螢光光譜和多維核磁共振等生物物理學方法來描繪 S100A13與amlexanox的交互作用，這些數據指出amlexanox可在非典型運輸途徑中阻絕S100A13與FGF1的結合。因此amlexanox可作為FGF1非典型運輸途徑拮抗劑的理由是藉由結合於S100A13與其交互作用對象的介面，導致強烈的抑制FGF1的非典型運輸途徑。此結果顯示amlexanox專一性的結合在S100A13上的FGF1結合區域。此二元複合體的水溶液結構有助於改進目前的拮抗劑或是設計出針對S100A13與FGF1更好的拮抗劑。 在第三章中，我們解出了S100A13-RAGE C2四維體的水溶液結構，並且其中S100A13-RAGE C2複合體中的結合區域有助於藥物開發。我們利用等溫滴定量熱法及1H-15N-HSQC 滴定來決定其結合區域，並利用多維核磁共振來解出S100A13-RAGE C2複合體結構，且深入了解其蛋白交互作用，此結果也證明S100A13與RAGE C2主要以疏水性作用力結合。由文獻中得知，藉由不同細胞種類與濃度，S100蛋白與RAGE的結合可誘發細胞增生或者凋亡。有趣的是，此二類細胞訊息均由ROS(reactive oxygen species)的形成來調控，並且由ROS在細胞中之持續時間來決定訊息。此研究中我們證明了探討S100A13-RAGE C2之交互作用有助於了解在S100蛋白與RAGE結合後所造成兩種不同細胞訊息傳導的形成。

碩士<br>中華醫事科技大學<br>生物科技系暨生物醫學研究所<br>102<br>Mounting evidence indicates that advanced glycation end-products (AGE) and their receptors (RAGE) have been implicated in tumor development and metastatic progression. However, prospective data on their association with cancer of specific sites, including colorectal cancer, are limited. We examined here whether and how AGE/RAGE could regulate cell growth in human colorectal adenocarcinoma cell lines (HT-29 and SW-480). We found that exposure of HT-29 and SW-480 cells to AGE (but not non-glycated BSA) increased the cell growth in concentration- and time-dependent manners. There were no changes in cytotoxicity in BSA or AGE treatment in these cells. Moreover, the JAK2-STAT1 signaling pathway was markedly activated by AGE in human colorectal cancer cells. The specific JAK2 inhibitor AG490 significantly prevented the proteins phosphorylation of JAK2 and STAT1 and reduced the effect of AGE-accelerated cellular growth. We also found that anti-RAGE antibody decreased the phosphorylation of JNK and p38 MAPK proteins and blocked the effect of AGE-induced cellular proliferation. These results suggest that AGE/RAGE has potent stimulatory effect on colorectal cancer cell growth, and the JAK2-STAT1 and the JNK-p38 MAPK signaling pathways can thus be considered as possible targets for colorectal cancer therapy.

碩士<br>國立清華大學<br>化學系<br>104<br>Abstract S100A12 (Calgranuline C) is a small and dimeric protein which belongs to the S100 family. When calcium ions bind to the two EF-hands of S100A12, the protein structure changes and promotes the interaction with its target proteins. RAGE (receptor for advanced glycation end) is one of the target protein for S100A12. By the past research, it showed the binding between these two proteins generated a pro-inflammatory response and activated several signal pathways such as ERK and NF-κB. It is an effective way that preventing the formation of S100A12-RAGE protein complex to inhibit various cancers. Tranilast, an anti-allergic drug that is usually used as treatment of allergic disorders, was found to have a strong interaction with S100A12 which may be a potential inhibitor. In this study, we discussed the interaction of S100A12 with RAGE and S100A12 with tranilast by their three dimension structure. We utilized several biophysical techniques, including multidimensional NMR spectroscopy, fluorescence spectroscopy, HADDOCK and WST-1 assay to characterize the structural information of protein complex. This study describes structural properties of S100A12-tranilast and S100A12-RAGE complex. It shows that tranilast blocks the binding sites of S100A12- RAGEV. This result can be useful for the development of new drug against various diseases.

碩士<br>國立陽明大學<br>醫學生物技術研究所<br>95<br>Receptor for advanced glycation end products (RAGE) is related with many clinical diseases such as diabetes, Alzheimer’s disease, chronic inflammation, and the metastasis of malignant tumors. Pituitary gland is located outside the blood brain barrier (BBB) and can be directly influenced by LPS in the blood stream during infection. We found that the folliculo-stellate (FS) like cells, TtT/GF cells expressed RAGE which involved in the expression of TNF-alpha, IL-1 beta and MIP-2 gene induced by LPS. However, the mechanism of RAGE in pituitary FS cells is still unclear. In present studies, we were interested in the RAGE-associated proteins and the mechanisms of these molecular. Our study employed three strategies to study RAGE-associated proteins. The first part was to predict RAGE associated proteins from four databases such as KinasePhos, HPRD, GPS, NetPhosK. There are 18 predicting candidates in these databases, such as CKI, CKII, PKC, PKG, ATM, IKK and so on. The second part was analyzing RAGE associated proteins by co-immunoprecipitation (co-IP). RAGE and its interacting proteins were precipitated together and separated by SDS-PAGE. After silver stain, the bands were analyzed by LC/MS. Because the low expression of RAGE, we tried to overexpress the RAGE cytosolic domain fusion protein to perform the analysis of LC/MS. We amplified the sequence of RAGE cytosolic domain and subcloned into pGEX-4T2 vector, and then produced the GST-CR43 fusion protein by E. coli BL21. The fusion protein was eluted by glutathione-S transferase (GST) pull down assay. The eluting proteins were separated by SDS-PAGE, and then the gel was stained with coomassie blue and silver stain. We picked the different bands to progress the analysis of LC/MS. A lot of proteins were identified, including many house-keeping proteins. The third part, we analyzed the interaction between RAGE co-IP product and the proteins that related with the RAGE downstream pathway in previous studies, for example ERK, JNK, and p38. The result revealed that RAGE interacted with p38, but the p38 binding site on RAGE cytosolic domain is still unknown. We also took shuttle vector pFlag-CMV2 to construct the plasmid expressing fusion protein flag-RAGE. We used the expression system to confirm the previous results. In the future, we will try to find the binding sites of RAGE interacting proteins using a series of C-terminal deletion mutants of human RAGE.

Chronic kidney disease (CKD) and acute kidney injury (AKI) are major public health problems. It is important to be able to identify those at high risk of adverse outcome, CKD progression and associated cardiovascular disease. The aim of the thesis was to study novel promising biomarkers, their relationship to kidney function, chronic inflammation and/or cardiovascular risk - placental growth factor (PlGF), pregnancy associated plasma protein A (PAPP-A), matrix metalloproteinase 2 (MMP-2), matrix metalloproteinase 9 (MMP-9), soluble receptor for advanced glycation end products (sRAGE), calcium binding protein S100A12 or extracellular newly identified RAGE binding protein (EN-RAGE), and high mobility group box protein-1 (HMGB-1) in patients with renal diseases including CKD, haemodialysis (HD), AKI patients, and healthy controls for comparison. First study revealed that PlGF is elevated in patients with decreased renal function. Second study demonstrated the association of MMP-2 and PAPP-A with proteinuria in patients with CKD. Moreover, serum MMP-2, MMP-9 and PAPP-A levels significantly differed in patients with various nephropathies. EN-RAGE levels are not elevated in patients with CKD, but are related to inflammatory status. PAPP-A, EN-RAGE and HMGB-1 levels are significantly elevated, but sRAGE and PlGF...