Introdução: Diabetes Mellitus é caracterizada por hiperglicemia crônica, e este aumento excessivo de glicose circulante pode gerar danos vasculares e microvasculares pela deposição de produtos de gliclação avançada (AGE), principalmente em estruturas com alta vascularização, como é o caso da sinóvia. Por todas estas razões, o presente estudo estabeleceu, de maneira temporal, o processo de acomentimento sinovial, através do grau de remodelamento e as proteínas envolvidas neste processo, tido como o gatilho na lesão da articulação do joelho. Foram utilizados ratos wistar (n=60), divididos em três grupos, conforme tempo de indução ( 7, 30 e 60 dias), cada grupo era composto de 10 animais diabéticos, induzido por estreptozotocina (35mg/kg de peso) e 10 animais controle, recebendo infusão do mesmo volume de solução salina, após o tempo estipulado os animais foram sacrificados e a sinóvia coletada para as análises propostas. Análise morfológica através de colorações de hematoxilina-eosina para análise do perfil celular do tecido sinovial e Picrosírius para avaliação da histoarquitetura das fibras colágenas. A quantificação das fibras colágenas foi realizada pela coloração do Picrosírius em microscópio de luz polarizada e a caracterização e distribuição de seus tipos por imunofluorescência, para quantificação total da proteina de colágeno foi realizado a medição da 4-hidroxiprolina (HPO). Os produtos de glicação avançada foram analisados e quantificados por imufluorescência. A detecção e quantificação da imunoexpressão de marcadores bioquímicos como ET-1, TGF-B e IL17 foi realizado por método estereológico de contagem de pontos em reticulo, e como método de confirmação dos achados imunohistoquimicos foi realizado análise de expressão gênica dos Colágenos I,III, e V alfa- 1, alfa-2), por Reação de Transcrição Reversa com amplificação por PCR em Tempo Real (qRT-PCR). Resultados: Foi observado modificação da estrutura sinovial de forma temporal, acometendo inicialmente os vasos subsinoviais e tecidos adjacentes a ele, isso foi observado em tanto em análise morfológica como confirmado em quantificação por Picro em luz polarizada, as modificações se mostraram significantes nos grupos de 30 e 60 dias, quando comparado ao respectivo grupo controle, houve aumento do colágeno total, através do Picrosirius, como por dosagem de HOP. Os resultados foram confirmados por imunofluorescência com o aumento progressivo do COLI e diminuição de COLIII e COLV, o RAGE e AGE também tiveram sua expressão aumentada conforme a evolução no tempo de indução dos animais. Em análise da expressão de outras proteínas foi possível observar a detecção da ET-1 e da IL-17 nos animais diabéticos em comparação ao controle, houve também expressão significativa do TGF-B quando comparado ao respectivo controle. Na análise da expressão gênica foi possível observar aumento do COLV inicialmente, principalmente da cadeia alfa 2, do COLIII e COLI, confirmando achados histomorfométricos. Conclusão: O tecido sinovial demonstra remodelamento precoce ao redor dos vasos, essa mediação envolve o COL1 e os produtos de glicação avançada. Esta alteração no tecido sinovial pode ser responsável por desencadear o acometimento articular no diabetes mellitus / Introduction: Diabetes Mellitus is characterized by chronic hyperglycemia, and this excessive increase of circulating glucose can cause vascular and microvascular damage by the deposition of advanced glycation products (AGE), especially in structures with high vascularization, as is the case of synovium. For all these reasons, the present study established, in a temporal way, the process of synovial concomitance, through the degree of remodeling and the proteins involved in this process, considered as the trigger in the lesion of the knee joint. Wistar rats (n = 60), divided into three groups, according to induction time (7, 30 and 60 days), each group consisted of 10 diabetic animals, induced by streptozotocin (35 mg / kg body weight) and 10 animals control, receiving infusion of the same volume of saline solution, after the stipulated time the animals were sacrificed and the synovium collected for the proposed analyzes. Morphological analysis using hematoxylineosin staining for analysis of the cellular profile of the synovial tissue and Picrosírius for evaluation of the histoarchitecture of the collagen fibers. The quantification of the collagen fibers was performed by the Picrosírius staining in a polarized light microscope and the characterization and distribution of its types by immunofluorescence, the measurement of 4-hydroxyproline (HPO) was performed for the total quantification of the collagen protein. Advanced glycation products were analyzed and quantified by impuluorescence. The detection and quantification of the immunoexpression of biochemical markers such as ET- 1, TGF-B and IL17 was performed by stereological method of reticule dot counting, and as a method of confirming the immunohistochemical findings, the analysis of the collagen I, III , and V alpha-1, alpha-2), by Reverse Transcription Reaction with Real-Time PCR Amplification (qRT-PCR). Results: Modification of the synovial structure was observed temporally, initially affecting subsynovial vessels and tissues adjacent to it, this was observed in both morphological analysis and confirmed in quantification by Picro in polarized light, the modifications were significant in the groups of 30 and 60 days, when compared to the respective control group, there was increase of the total collagen, through Picrosirius, as per HOP dosage. The results were confirmed by immunofluorescence with progressive increase of COLI and decrease of COLIII and COLV, RAGE and AGE also had their expression increased as the evolution in the induction time of the animals. In the analysis of the expression of other proteins it was possible to observe the detection of ET-1 and IL-17 in diabetic animals in comparison to the control, there was also significant expression of TGF-B when compared to the respective control. In the analysis of the gene expression it was possible to observe an increase of the COLV initially, mainly of the alpha 2 chain, of the COLIII and COLI, confirming histomorphometric findings. Conclusion: Synovial tissue demonstrates early remodeling around vessels, this mediation involves COL1 and advanced glycation products. This change in synovial tissue may be responsible for triggering joint involvement in diabetes mellitus

A glicação não enzimática das proteínas é um fator comum para a fisiopatologia de uma série de transtornos relacionados ao envelhecimento e a doenças como o diabetes mellitus (DM). O geração dos produtos de glicação, os AGEs (do inglês: Advanced Glycation End Products) se dá através de reações de glicação da mariz extracelular (MEC) na derme e têm sido apontado como um dos fatores responsáveis pela perda de elasticidade e deficiência de cicatrização da pele. A permeação cutânea de compostos anti-AGE é uma limitação importante para eficiência terapêutica de compostos que devem atingir camadas mais profundas da pele. Modelos de pele reconstruída contendo equivalente dérmico glicado são estruturas tridimensionais geradas in vitro que mimetizam a pele humana e representam um eficiente modelo para o estudo de células e modificações provocadas na MEC no processo de envelhecimento e DM. O modelo 3D de pele reconstruída tem características metabólicas, de permeabilidade e atividade semelhantes à da pele original, potencializando seu papel nas investigações sobre permeabilidade de drogas, toxicidade, irritação, eficácia e segurança de compostos e diferenciação de queratinócitos. Uma série de compostos naturais ou sintéticos inibidores de AGEs têm sido descobertos e apresentados recentemente e podem representar inovação terapêutica no tratamento de modificações causadas pela a formação e acúmulo destes AGEs também na pele. Este estudo avaliou o desenvolvimento da pele reconstruída glicada e posteriormente, a avaliação da eficácia e toxicidade de compostos anti-glicação como aminoguanidina e carnosina em modelo de pele reconstruída glicada. Em perspectiva, este estudo contribuiu para o desenvolvimento de uma nova tecnologia in vitro, a pele reconstruída glicada, que auxiliará a compreensão da biologia da interação célula-MEC mimetizando processos fisiopatológicos importantes como o envelhecimento e o DM. / The Advanced Glycation End Products (AGEs) of proteins is a common factor to the pathophysiology of a number of disorders related to aging and diseases such as diabetes mellitus (DM). The generation of the AGEs products on skin occurs mainly through non-enzymatic glycation reactions of the dermal extracellular matrix and has been touted as one of the factors responsible for loss of elasticity and disability of skin healing. The skin permeation of compounds is an important limitation for therapeutic/cosmetic efficacy of anti-AGE compounds, which must reach the deepest layers of the skin. Reconstructed skin model containing dermal equivalent modified by in vitro glycation is able to mimic the elderly human skin and represent an efficient model for the study of cells interactions and changes in extracellular matrix induced by aging and diabetes. The 3D reconstructed skin model has metabolic characteristics, permeability and activity similar to the original skin, reinforcing its role in drug permeability of investigations toxicity, irritation, safety and efficacy evaluation of compounds and differentiation of keratinocytes. A number of natural or synthetic AGEs inhibitor compounds have been recently discovered and displayed and can represent therapeutic innovation for the treatment of changes caused by the aging of the skin. In this study we performed the development of reconstructed glycated skin model and evaluated the efficacy and toxicity of anti-glycation compounds such as aminoguanidine and carnosine. In perspective, this study has contributed to the development of a new technology in vitro, and for the understanding cell-extracellular matrix interaction during the aging of skin.

Diabetes is a chronic disease with high prevalence and significant morbidity. Chronic changes in the wall of small and large vessels lead to main diabetes complications. Apart from long- term hyperglycemia, several factors are involved in the development of diabetes vasculopathy. The aim of this work was to describe new early biomarkers of these vascular changes, to identify risky patients. Alongside, gene polymorphisms involved in protective pathways of glucose metabolism were studied. In three human studies with Type 1 (T1D) and Type 2 (T2D) diabetes patients special biochemical parameters of receptor for advanced glycation endproducts (RAGE) and its ligands, deglycation enzyme glyoxalase 1 (GLO1) and fructosamine 3-kinase (FN3K) gene polymorphisms were analyzed. Non-invasive measurement of glycation by skin autofluorescence (SAF) was assessed in all subjects. Soluble RAGE, HMGB1 and endothelial dysfunction markers were increased in patients with diabetes as compared with controls, however the differences between T1D and T2D were not significant. For the first time, an association between FN3K (rs1056534) and (rs3848403) polymorphism and sRAGE concentration in diabetes was shown. GLO1 (rs4746) polymorphism was associated with changes in endothelial dysfunction. Patients with diabetes had higher...

Sowohl bei der Forschung der Maillard-Reaktion in Lebensmitteln als auch im menschlichen Körper lag das Hauptaugenmerk bislang auf der Reaktion der Carbonylfunktion mit den Aminofunktionen der Seitenketten wie Lysin oder Arginin, da sie in vielen Lebensmitteln oder physiologischen Proteinen die größte Quelle an Aminofunktionen darstellen. Dagegen wurde eine vergleichbare Reaktion mit dem N-Terminus von Aminosäuren, Peptiden oder Proteinen weniger beachtet, obgleich in lysinarmen oder peptidhaltigen Lebensmitteln die N-terminalen Aminofunktionen dominieren und die Seitenketten körpereigener Proteine räumlich für einen Angriff der Carbonylfunktion unzugänglich sein können. Da in den HA-Nahrungen die allergieauslösenden Proteine hydrolytisch gespalten vorliegen, stehen für eine mögliche Amadori-Produktbildung gegenüber den konventionellen Säuglingsnahrungen quantitativ mehr alpha-Aminogruppen als epsilon-Aminogruppen zur Verfügung. Demzufolge sollte für die Beurteilung von HA-Nahrungen eine Methode entwickelt werden, mit deren Hilfe eine Aussage über die Amadori-Produktbildung auch am N-Terminus getroffen werden kann. Aufbauend auf den Ergebnissen der Furoylmethylderivate-Bestimmung (FMAA-Bestimmung) in peptidhaltigen Lebensmitteln, war es ein weiteres Ziel der vorliegenden Dissertation die entwickelte Methode auch auf ihre Anwendbarkeit auf die Beurteilung des Glykierungsstatus des Hämoglobin in vivo zu testen. Nach der N-terminalen Amadori-Produktbildung im Zuge der frühen Phase der Maillard-Reaktion lag im zweiten Teil der Dissertation das Hauptaugenmerk auf die fortgeschrittene Phase der Maillard-Reaktion am N-Terminus von Peptiden oder Proteinen. Der Schwerpunkt lag dabei auf der Bildung von 2-(1H)-Pyrazinonen im komplexen trockenen Lebensmittel und in vivo.

Peritoneal dialysis (PD) is a form of renal replacement therapy using the peritoneum as a dialysis membrane. PD solutions employed to remove nitrogen metabolites and excess plasma fluid, and to restore electrolyte and acid-base balance are being developed to minimize local and systemic inflammatory responses while maintaining peritoneal homeostasis and host defense. The effect of chronic action of PD solutions on the peritoneum results in its remodeling and, possibly, eventual loss of peritoneal ultrafiltration capacity. Factors most responsible for late complications and peritoneal remodeling include high glucose levels in PD solutions, and the presence and formation of glucose degradation products (GDP) and advanced glycation end - products (AGEs) in the peritoneal cavity. The aim of our study described in this dissertation was to test various PD solutions with different glucose content and GDP and, using AGEs receptor ligands, to define their systemic effects and identify PD solutions with highest biocompatibility. This part of the dissertation characterizes conventional glucose - based solutions, low - glucose and GDP load solutions as well as glucose polymer (icodextrin) - based PD solutions while determining the plasma and dialysate levels of soluble receptor for AGEs (s - RAGE) and its...

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The reactive 1,2-dicarbonyl compound methylglyoxal (MGO) is consumed with food and its concentrations decrease during digestion. In the present paper, the reaction of MGO with creatine, arginine, and lysine during simulated digestion, and its reaction with creatine during the digestion in human volunteers, was studied. Therefore, simulated digestion experiments with a gastric and an intestinal phase were performed. Additionally, an intervention study with 12 subjects consuming MGO-containing Manuka honey and creatine simultaneously or separately was conducted. Derivatization with o-phenylenediamine and HPLC–UV was used to measure MGO, while creatine and glycated amino compounds were analyzed via HPLC–MS/MS. We show that MGO quickly reacts with creatine and arginine, but not lysine, during simulated digestion. Creatine reacts with 56% of MGO to form the hydroimidazolone MG-HCr, and arginine reacted with 4% of MGO to form the hydroimidazolone MG-H1. In the intervention study, urinary MG-HCr excretion is higher in subjects who consumed MGO and creatine simultaneously compared to subjects who ingested the substances separately. This demonstrates that the 1,2-dicarbonyl compound MGO reacts with amino compounds during human digestion, and glycated adducts are formed. These contribute to dietary glycation products consumed, and should be considered in studies investigating their physiological consequences.

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.

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.

A glicação é uma reação não enzimática que ocorre entre proteínas e açúcares redutores e, é responsável pela formação de adultos e de ligações cruzadas entre proteínas, como por exemplo: a pentosidina, produto final de glicação avançada que se acumula em vários tecidos ao longo do tempo. A glicação é deletéria para o organismo e está associada a modificações estruturais em proteínas e alterações de suas funções específicas, tais como: atividade enzimática, capacidade de ligação e tempo de vida de proteínas, além de ser responsável pela produção de espécies reativas de oxigênio (EROS). O mecanismo de formação da pentosidina envolve reações oxidativas e, uma das estratégias para minimizá-Ia é o aumento da atividade antioxidante nos tecidos. A pariparoba (Pothomorphe umbellata (L.) Miq) demonstrou atividade antioxidante in vitro e in vivo quando aplicada sobre a pele. Essa atividade foi atribuída ao 4-nerolidilcatecol (4-NC), que se mostrou 10 vezes mais potente que o α-tocoferol. Os extratos de pariparoba também inibiram a lipoperoxidação espontânea da pele em camundongos sem pelo. Neste trabalho empregou-se o modelo de glicação de albumina de soro bovino (BSA) frente à D-ribose, com avaliação da fluorescência produzida pela pentosidina formada na reação. Avaliou-se igualmente a atividade do 4-NC em diferentes concentrações sobre a reação de glicação da BSA em presença de D-ribose após 24 horas, empregando-se a aminoguanidina como controle positivo. Nas condições experimentais o 4-NC não foi capaz de inibir a reação de glicação, ao contrário da aminoguanidina. Foi também utilizado modelo para avaliação da propriedade contrátil de fibroblastos em matriz tridimensional de gel de colágeno, glicado e não glicado com D-ribose. O 4-NC na concentração de 100 µM permitiu a manutenção da propriedade contrátil de fibroblastos em gel colágeno glicado. Estudos de glicação em maiores períodos de tempo devem ser realizados visando a confirmar a possível atividade anti-glicação deste composto. / Glycation is a non enzymatic reaction which occurs between proteins and reductor sugars, responsible for the formation of adducts and crosslinkers between proteins, such as, pentosidine, an advanced glycation end-product (AGE) which accumulates in many tissues during aging. AGEs accumulation is deleterious to the body and is associated with structural modifications in proteins and imbalance in their specific functions, such as: enzymatic activity, binding capacity, protein turnover and also responsible for the production of reactive oxygen species (ROS). The mechanism of pentosidine formation involves oxidative reactions. One of the strategies to reduce pentosidine formation is by increasing antioxidant activity in tissues. Pariparoba (Pothomorphe umbellata (L.) Miq. has showed antioxidant activity in vitro and in vivo when applied on the skin. This activity was attributed to 4-nerolydilcatechol (4-NC), which is 10 times more potent than α-tocopherol. Extracts of Pariparoba also inhibited the spontaneous lipid peroxidation in the skin of hairless mice. In this work, the bovine serum albumin (BSA) model for glycation with D-ribose, evaluated by pentosidine fluorescence spectroscopy was employed. The activity of 4¬NC was evaluated in different concentrations in this model after 24 hours. Aminoguanidine was used as positive control. In this experimental condition, 4-NC was not capable to inhibit the BSA glycation. We also evaluated the contractile properties of fibroblasts on tridimensional matriz of collagen gel glycated or not with D-ribose. 4-NC (100 µM) was able to keep the contractile capacity of fibroblasts in glycated collagen. Studies of glycation in longer periods of time should be made in order to further evaluate the possible anti-glycation activity of this compound.