The principal cause of non-ST-segment myocardial infarction (NSTEMI), a subclass of acute coronary syndrome (ACS), is thrombosis and the underlying cause is atherosclerosis. Percutaneous coronary intervention (PCI) is one of the treatments to attenuate the ischemic effects of severe coronary artery stenosis. However, restenosis following PCI (post-PCI) is a major problem for the long-term success of the procedure. Recently, the interaction of advanced glycation end products (AGE) with the receptor for advanced glycation end products (RAGE) has been implicated in the development of atherosclerosis in animal models. Interaction of AGE with RAGE results in activation of nuclear factor kappa-B (NF-êB), release of cytokines including tumor necrosis factor-alpha (TNF-á), the expression of adhesion molecules including soluble vascular adhesion molecule-1 (sVCAM-1) and induction of oxidative stress all of which have been implicated in the development of atherosclerosis. The soluble receptor for advanced glycation end products (sRAGE) acts as a decoy for RAGE ligands (AGEs) and this occurs by competing with RAGE. In animal models, balloon inflation and de-endothelialization of the carotid artery increase the concentration of AGE and RAGE in the arterial wall and induces neointimal hyperplasia and stenosis. Treatment with sRAGE in animal models reduces neointimal growth and decreases smooth muscle cell migration and proliferation and expression of extracellular matrix.<p> It is hypothesized that NSTEMI and post-PCI restenosis may be due to low levels of serum sRAGE resulting in increased AGE and RAGE interactions. Low levels of sRAGE would also increase the levels of serum TNF-á and sVCAM-1.<p> The objectives of this study were to determine whether: (1) the levels of serum sRAGE are lower and the levels of AGE, TNF-á and sVCAM-1 are higher in NSTEMI patients compared to control; (2) the levels of serum sRAGE are lower and the levels of AGE, TNF-á and sVCAM-1 are higher in NSTEMI patients with restenosis compared to those without restenosis; and (3) sRAGE or AGE/sRAGE ratio may serve as a biomarker/ predictor of NSTEMI and post-PCI restenosis.<p> The study objectives include 46 consecutive NSTEMI patients undergoing elective PCI and 28 healthy age-matched male controls. Pre-PCI and 6 month post-PCI angiography were performed in all NSTEMI patients. Blood samples were collected at designated intervals for the measurement of sRAGE, AGE, TNF-á, and sVCAM-1 using commercially available enzyme-linked immunosorbent assay (ELISA) kits.<p> The levels of serum sRAGE were lower and those of TNF-á, sVCAM-1, AGE and AGE/sRAGE were higher in NSTEMI patients compared to control subjects. (sRAGE, 884.55 ± 50 vs. 1287 ± 41.5 pg/mL{p<0.001}; TNF-á, 23.1 ± 2.3 vs. 10.3 ± 0.8 pg/mL {p<0.002}; sVCAM-1, 1059.62 ± 70.8 vs. 651 ± 35.5 ng/mL {p<0.0003}, AGE, 1192.50 ± 82.6 vs. 669.40 ± 47.9 ng/mL {p<0.001}; and AGE/sRAGE, 1.75 ± 0.17 vs. 0.52 ± 0.06 {p<0.001}).<p> The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy of the sRAGE biomarker test were 59%, 100%, 100%, 100% and 74% respectively, while those of the AGE/sRAGE test were 85%, 91%, 97%, 67% and 86%, respectively, in the diagnosis of patients with NSTEMI acute coronary syndrome. The pre-PCI levels of serum sRAGE in patients with restenosis were significantly lower (p<0.001) than in patients without restenosis (610.6 ± 24.1 vs. 1143.8 ± 52.5 pg/mL). The post-PCI levels of serum sRAGE were significantly lower (p<0.0001) in patients with restenosis compared to those without restenosis (477 ± 18.6 vs. 1106.7 ± 41.9 pg/mL). The pre-PCI levels of serum TNF-á and sVCAM-1 were significantly higher (p<0.001 and p<0.001) in patients with restenosis compared to those without restenosis (TNF-á, 37.9 ± 2.5 vs. 11.6 ± 0.41 pg/mL; sVCAM-1, 1381.8 ± 63.5 vs. 811.37 ± 26.5 ng/mL). The post PCI levels of serum TNF-á and sVCAM-1 were also significantly higher (p<0.0001 and p<0.0001) in patients with restenosis than in patients without restenosis (TNF-á, 48.4 ± 1.4 vs. 12.5 ± 0.44 pg/mL; sVCAM-1, 1381.8 ± 63.5 vs. 762.2 ± 26.4 ng/mL). The post-PCI levels sRAGE were lower while those of TNF-á and sVCAM-1 were higher compared to the pre-PCI levels in patients with restenosis. However, the pre- and post-PCI levels of serum sRAGE, TNF-á and sVCAM-1 were similar in patients without restenosis. The pre-PCI levels of serum AGE and AGE/sRAGE were significantly higher (p<0.001 and p<0.001) in patients with restenosis compared to those without restenosis (AGE, 1512.1 ± 84.53 vs. 891.7 ± 92.4 ng/mL; 2.39 ± 0.20 vs. 1.03 ± 0.17).<p> The sensitivity, specificity, PPV, NPV, and accuracy of the pre-PCI sRAGE tests were 73%, 100%, 100%, 80%, and 87%, respectively while those of the AGE/sRAGE tests were 81%, 94%, 93%, 84% and 88%, respectively in identifying patients with post-PCI restenosis.<p> In conclusion the results suggest that: (1) the levels of serum sRAGE are lower while those of TNF-á, sVCAM-1, AGE and AGE/sRAGE are higher in NSTEMI patients compared to control subjects; (2) serum levels of sRAGE are negatively correlated with the number of diseased vessels; (3) Both low sRAGE and high AGE/sRAGE may serve as a biomarker/predictor of NSTEMI, but AGE/sRAGE has a greater sensitivity compared to sRAGE; (4) the pre-PCI levels of serum sRAGE are lower while those of AGE/sRAGE are higher in patients with restenosis compared to those without restenosis; and (5) both low sRAGE and high AGE/sRAGE may serve as a predictor/ biomarker of post-PCI restenosis; however, AGE/sRAGE has a greater sensitivity than sRAGE.

The principal cause of non-ST-segment myocardial infarction (NSTEMI), a subclass of acute coronary syndrome (ACS), is thrombosis and the underlying cause is atherosclerosis. Percutaneous coronary intervention (PCI) is one of the treatments to attenuate the ischemic effects of severe coronary artery stenosis. However, restenosis following PCI (post-PCI) is a major problem for the long-term success of the procedure. Recently, the interaction of advanced glycation end products (AGE) with the receptor for advanced glycation end products (RAGE) has been implicated in the development of atherosclerosis in animal models. Interaction of AGE with RAGE results in activation of nuclear factor kappa-B (NF-êB), release of cytokines including tumor necrosis factor-alpha (TNF-á), the expression of adhesion molecules including soluble vascular adhesion molecule-1 (sVCAM-1) and induction of oxidative stress all of which have been implicated in the development of atherosclerosis. The soluble receptor for advanced glycation end products (sRAGE) acts as a decoy for RAGE ligands (AGEs) and this occurs by competing with RAGE. In animal models, balloon inflation and de-endothelialization of the carotid artery increase the concentration of AGE and RAGE in the arterial wall and induces neointimal hyperplasia and stenosis. Treatment with sRAGE in animal models reduces neointimal growth and decreases smooth muscle cell migration and proliferation and expression of extracellular matrix.<p> It is hypothesized that NSTEMI and post-PCI restenosis may be due to low levels of serum sRAGE resulting in increased AGE and RAGE interactions. Low levels of sRAGE would also increase the levels of serum TNF-á and sVCAM-1.<p> The objectives of this study were to determine whether: (1) the levels of serum sRAGE are lower and the levels of AGE, TNF-á and sVCAM-1 are higher in NSTEMI patients compared to control; (2) the levels of serum sRAGE are lower and the levels of AGE, TNF-á and sVCAM-1 are higher in NSTEMI patients with restenosis compared to those without restenosis; and (3) sRAGE or AGE/sRAGE ratio may serve as a biomarker/ predictor of NSTEMI and post-PCI restenosis.<p> The study objectives include 46 consecutive NSTEMI patients undergoing elective PCI and 28 healthy age-matched male controls. Pre-PCI and 6 month post-PCI angiography were performed in all NSTEMI patients. Blood samples were collected at designated intervals for the measurement of sRAGE, AGE, TNF-á, and sVCAM-1 using commercially available enzyme-linked immunosorbent assay (ELISA) kits.<p> The levels of serum sRAGE were lower and those of TNF-á, sVCAM-1, AGE and AGE/sRAGE were higher in NSTEMI patients compared to control subjects. (sRAGE, 884.55 ± 50 vs. 1287 ± 41.5 pg/mL{p<0.001}; TNF-á, 23.1 ± 2.3 vs. 10.3 ± 0.8 pg/mL {p<0.002}; sVCAM-1, 1059.62 ± 70.8 vs. 651 ± 35.5 ng/mL {p<0.0003}, AGE, 1192.50 ± 82.6 vs. 669.40 ± 47.9 ng/mL {p<0.001}; and AGE/sRAGE, 1.75 ± 0.17 vs. 0.52 ± 0.06 {p<0.001}).<p> The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy of the sRAGE biomarker test were 59%, 100%, 100%, 100% and 74% respectively, while those of the AGE/sRAGE test were 85%, 91%, 97%, 67% and 86%, respectively, in the diagnosis of patients with NSTEMI acute coronary syndrome. The pre-PCI levels of serum sRAGE in patients with restenosis were significantly lower (p<0.001) than in patients without restenosis (610.6 ± 24.1 vs. 1143.8 ± 52.5 pg/mL). The post-PCI levels of serum sRAGE were significantly lower (p<0.0001) in patients with restenosis compared to those without restenosis (477 ± 18.6 vs. 1106.7 ± 41.9 pg/mL). The pre-PCI levels of serum TNF-á and sVCAM-1 were significantly higher (p<0.001 and p<0.001) in patients with restenosis compared to those without restenosis (TNF-á, 37.9 ± 2.5 vs. 11.6 ± 0.41 pg/mL; sVCAM-1, 1381.8 ± 63.5 vs. 811.37 ± 26.5 ng/mL). The post PCI levels of serum TNF-á and sVCAM-1 were also significantly higher (p<0.0001 and p<0.0001) in patients with restenosis than in patients without restenosis (TNF-á, 48.4 ± 1.4 vs. 12.5 ± 0.44 pg/mL; sVCAM-1, 1381.8 ± 63.5 vs. 762.2 ± 26.4 ng/mL). The post-PCI levels sRAGE were lower while those of TNF-á and sVCAM-1 were higher compared to the pre-PCI levels in patients with restenosis. However, the pre- and post-PCI levels of serum sRAGE, TNF-á and sVCAM-1 were similar in patients without restenosis. The pre-PCI levels of serum AGE and AGE/sRAGE were significantly higher (p<0.001 and p<0.001) in patients with restenosis compared to those without restenosis (AGE, 1512.1 ± 84.53 vs. 891.7 ± 92.4 ng/mL; 2.39 ± 0.20 vs. 1.03 ± 0.17).<p> The sensitivity, specificity, PPV, NPV, and accuracy of the pre-PCI sRAGE tests were 73%, 100%, 100%, 80%, and 87%, respectively while those of the AGE/sRAGE tests were 81%, 94%, 93%, 84% and 88%, respectively in identifying patients with post-PCI restenosis.<p> In conclusion the results suggest that: (1) the levels of serum sRAGE are lower while those of TNF-á, sVCAM-1, AGE and AGE/sRAGE are higher in NSTEMI patients compared to control subjects; (2) serum levels of sRAGE are negatively correlated with the number of diseased vessels; (3) Both low sRAGE and high AGE/sRAGE may serve as a biomarker/predictor of NSTEMI, but AGE/sRAGE has a greater sensitivity compared to sRAGE; (4) the pre-PCI levels of serum sRAGE are lower while those of AGE/sRAGE are higher in patients with restenosis compared to those without restenosis; and (5) both low sRAGE and high AGE/sRAGE may serve as a predictor/ biomarker of post-PCI restenosis; however, AGE/sRAGE has a greater sensitivity than sRAGE.

The doctoral dissertacion is focused on the role of inflammation in the pathogenesis of preterm labor. In the first part, we describe the current view on pathophysiology of preterm labor. In the second part, we evaluated the relationship of specific markers of inflammation and endothelial dysfunction to preterm birth and fetal inflammatory response. The most important findings of our study was that we found decreased levels of MMP-2 and decreased levels of sRAGE in women with preterm labor in comparison with the control group of pregnant women. Similarly, we found decreased levels of MMP-2 in women with subsequent diagnosed fetal inflammatory response. sRAGE is currently ranked among patttern recognition receptors. In the case of sRAGE we followed the results of our pilot project, it can be assumed that the its low level are connected with tissue damage. We confirmed that it can play an important role in the pathogenesis of preterm labor. We assume abnormal regulatory mechanisms of the production of MMP-2. In both cases, however, further studies are required to elucidate the functional significance of our results.

The doctoral dissertacion is focused on the role of inflammation in the pathogenesis of preterm labor. In the first part, we describe the current view on pathophysiology of preterm labor. In the second part, we evaluated the relationship of specific markers of inflammation and endothelial dysfunction to preterm birth and fetal inflammatory response. The most important findings of our study was that we found decreased levels of MMP-2 and decreased levels of sRAGE in women with preterm labor in comparison with the control group of pregnant women. Similarly, we found decreased levels of MMP-2 in women with subsequent diagnosed fetal inflammatory response. sRAGE is currently ranked among patttern recognition receptors. In the case of sRAGE we followed the results of our pilot project, it can be assumed that the its low level are connected with tissue damage. We confirmed that it can play an important role in the pathogenesis of preterm labor. We assume abnormal regulatory mechanisms of the production of MMP-2. In both cases, however, further studies are required to elucidate the functional significance of our results.

Les pathologies cardio-vasculaires (CVD) représentent la principale cause de morbidité et demortalité dans le monde. Le risque de CVD augmente avec l’âge, le tabagisme, le diabète, les dyslipidémies, l’obésité et l’insuffisance rénale. L’incidence et la prévalence des CVD nécessitent le développement de stratégies de prévention et de traitement, et la recherche de nouveaux biomarqueurs. Le récepteur aux produits de glycation avancée (RAGE) est impliqué dans plusieurs pathologies métaboliques ou inflammatoires. L’activation du RAGE par ses multiples ligands, i.e. produits de glycation avancée (AGE), protéines de la famille S100 et amphotérine (HMGB1) induit une cascade pro-inflammatoire. La forme soluble du RAGE (sRAGE) a été proposée comme biomarqueur du risque vasculaire, de la sévérité et du devenir des CVD, particulièrement chez les patients diabétiques ou insuffisants rénaux. Cependant, les données sont contradictoires et des corrélations positives et négatives sont observées pour une même pathologie. L’importance de l’axe ligand-RAGE dans les processus pathologiques et le large éventail de molécules se liant au RAGE (des protéines proinflammatoires aux auto-anticorps), justifient le présent travail. Au cours du présent travail, dans un premier temps, nous avons d’abord étudié les effets des ligands du RAGE et des auto-anticorps anti-sRAGE récemment décrit, sur la quantification du sRAGE en ELISA. Nous supposons que l’interaction entre le sRAGE et ces molécules pourrait perturber le dosage du sRAGE. Dans un deuxième travail, nous avons évalué les variations du taux de sRAGE et des auto-anticorps anti-sRAGE après chirurgie bariatrique d’une obésité morbide. Les ligands du RAGE (Nε-carboxyméthyllysine, S100A6, S100A12, S100B, HMGB1 et peptide β-amyloïde) se fixent au sRAGE en différents sites et pourraient potentiellement interférer dans sa quantification par l’intermédiaire d’un masquage d’épitope. Nous avons incubé ces ligands, à des concentrations physiologiques et pathologiques, avec du sRAGE recombinant et du sérum pour évaluer leur effet sur le dosage du sRAGE. Des auto-anticorps anti-sRAGE ont été identifiés et purifiés et leur effet sur le dosage de sRAGE a été évalué. La présence des ligands ou d’autoanticorps anti-sRAGE ne modifie pas le dosage du sRAGE recombinant ou sérique. L’obésité favorise les dyslipidémies, les perturbations glycémiques et l’inflammation, conditions au cours desquelles le RAGE pourrait jouer un rôle important. Nous avons étudié les variations des taux sériques du sRAGE et de ses auto-anticorps et leur évolution avec l’amélioration métabolique des sujets obèses après chirurgie bariatrique. Les patients ont été sélectionnés au sein d’une cohorte déjà établie (Patient présentant une obésité morbide et candidat à une chirurgie de bypass gastrique, ABOS, Lille). Les patients présentant des facteurs pouvant modifier les niveaux de sRAGE, tels qu’un traitement par statines, une insuffisance rénale chronique ou une hypertension,ont été exclus. Comparé au groupe contrôle, les taux de sRAGE et d’auto-anticorps étaientsignificativement plus élevés chez les patients obèses avant la chirurgie. Parallèlement à la baisse de l’indice de masse corporelle, les taux de sRAGE et d’anti-sRAGE ont été significativement diminués un an après la chirurgie. La baisse d’anti-sRAGE a été corrélée à l’augmentation des taux de HDL. Nous démontrons que les variations des taux de sRAGE constatées dans la littérature ne sont, à priori, pas dues à l’interaction des ligands du RAGE avec le sRAGE. D’autres hypothèses, comme la régulation de la formation et de la clairance du sRAGE, sont discutées. Nous avons, pour la première fois, démontré la présence d’auto-anticorps anti-sRAGE chez les patients obèses, et la diminution du taux de ces auto-anticorps après une chirurgie de bariatrique. Ces résultats suggèrent que l’obésité pourrait être responsable d’une réaction auto-immune contre le sRAGE. / 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 (N&#949;-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

Rheumatoid arthritis is a chronic inflammatory autoimmune disease. Measurement of the level of serum markers (sRAGE, S100A9, S100A8 and S100A12) and genetic testing for the presence of the PTPN22 genetic polymorphism could help elucidate the underlying cause of inflammation and complications in RA, such as atherosclerosis. Therefore, serum levels of sRAGE, S100A9, S100A8 and S100A12 were measured by ELISA in patients with established RA (n=138). The associations between the serum levels of these molecules; and inflammatory markers and RA complications were analysed by multiple linear regression modelling. Established RA patients (n=192) were investigated for the PTPN22 C1858T genetic polymorphism by PCR-RFLP. Multiple logistic regression modelling was used to examine the association between PTPN22 C1858T genetic polymorphism and inflammatory markers and RA complications. In RA patients, we found that serum levels of S100A9 were associated with the body mass index (BMI); and the presence of S100A8 and S100A12. The serum levels of S100A8 in RA patients were associated with the presence of anti-citrullinated peptide antibodies, rheumatoid factor and S100A9. The serum levels of S100A12 in RA patients were associated with the presence of anti-citrullinated peptide antibodies and S100A9; and a history of diabetes. Inflammatory markers and RA complications were not associated with the PTPN22 genetic polymorphism in established RA patients; serum level of triglyceride was the only variable associated with PTPN22 C1858T in multiple logistic regression analysis. Taken together, these data suggest that serum levels of sRAGE, S100A9 and S100A12 protein may be useful correlates of inflammation and autoantibody production in RA patients. Further studies are recommended to determine whether these markers predict clinical outcomes when measured at the onset of RA.

Submitted by Ana Lúcia Torres (bfmhuap@gmail.com) on 2018-01-10T14:41:48Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) CHRISTIANE BENSUSAN.pdf: 1975827 bytes, checksum: 4184e4e56bd63c7b405bc35f03852e9a (MD5) / Approved for entry into archive by Ana Lúcia Torres (bfmhuap@gmail.com) on 2018-01-10T14:41:57Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) CHRISTIANE BENSUSAN.pdf: 1975827 bytes, checksum: 4184e4e56bd63c7b405bc35f03852e9a (MD5) / Made available in DSpace on 2018-01-10T14:41:57Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) CHRISTIANE BENSUSAN.pdf: 1975827 bytes, checksum: 4184e4e56bd63c7b405bc35f03852e9a (MD5) Previous issue date: 2017 / UNIMED - Rio Empreendimentos Médicos e Hospitalares / Introdução: O Diabetes Mellitus tipo 2 (DM2) representa um importante problema de saúde pública, considerando sua grande morbi/mortalidade, decorrente das complicações crônicas da doença. A formação de produtos finais de glicação avançada (AGE) representa um dos diversos mecanismos fisiopatológicos implicados na gênese das complicações do DM2. Através da ligação dos AGE com os seus receptores teciduais (RAGE) vias intracelulares são ativadas, levando ao aumento da resposta inflamatória e à indução de estresse oxidativo, que culminam com as complicações micro e macrovasculares do DM2. Por outro lado, há um pool de RAGE solúveis (sRAGE), constituído por variantes do RAGE (esRAGE e cRAGE), com capacidade de interagir com os mesmos ligantes do RAGE tecidual, sem, entretanto, desencadear a transdução do sinal intracelular após a sua ligação. Dessa forma, o sRAGE funcionaria como um fator protetor para o desenvolvimento das complicações crônicas do DM2. No entanto, a associação entre os níveis de sRAGE com a presença de complicações micro e acrovasculares do DM2, assim como a associação entre os níveis de sRAGE com o grau de controle glicêmico, não está bem estabelecida, já que os trabalhos existentes na literatura mostram resultados divergentes. Objetivos: O presente estudo visou compreender melhor as correlações entre os níveis de sRAGE, as complicações crônicas do DM2 e o controle glicêmico, através da avaliação de pacientes DM2 acompanhados no Hospital Universitário Antônio Pedro. Métodos: Foram incluídos 89 pacientes DM2, 43 com complicações e 46 sem complicações. Cada um desses dois grupos pacientes foi subdividido, de acordo com o controle glicêmico, em outros 3 subgrupos. Os pacientes foram submetidos a uma avaliação clínica e laboratorial, com dosagem de sRAGE e hemoglobina glicada. O sRAGE foi mensurado no soro dos pacientes utilizando-se o teste imunoenzimático ELISA. Resultados: Não foi encontrada diferença estatisticamente significativa nos níveis de sRAGE plasmático entre os pacientes DM2 com e sem complicações microvasculares do DM2, bem como não foi constatada correlação do sRAGE com o controle glicêmico. Conclusão: Novos estudos são necessários para melhor elucidar os mecanismos envolvidos na produção e regulação da concentração das formas solúveis do RAGE e esclarecer a relação de causa-efeito entre os níveis séricos do sRAGE e as complicações crônicas do DM2 / Background: Type 2 diabetes mellitus (T2DM) is a major public health problem, considering its high morbidity and mortality due to chronic complications. The formation of advanced glycation end products (AGE) is one of several pathophysiological mechanisms involved in the development of complications of T2DM. By binding of AGE with its tissue receptors (RAGE), intracellular pathways are activated, leading to increased inflammatory response and the induction of oxidative stress, which culminate in the micro and macrovascular complications of T2DM. Moreover, there is a pool of soluble RAGE (sRAGE) consisting of variants of RAGE (esRAGE and cRAGE), with ability to interact with the same tissue RAGE ligands, without, however, trigger the intracellular signal transduction after binding. Thus, sRAGE would behave as a protective factor for the development of chronic complications of T2DM. Nevertheless, the association between sRAGE levels with the presence of micro and macrovascular complications of T2DM, as well as the association between sRAGE levels with the degree of glycemic control, is not well established, since the studies in the literature show divergent results. Objectives: This study aimed to better understand the correlations between the levels of sRAGE, the chronic complications of T2DM and glycemic control, through the evaluation of T2DM patients treated at University Hospital Antônio Pedro. Methods: A total of 89 T2DM patients were included, 43 with complications and 46 without complications. Each of these two groups was divided according to glycemic control, in other 3 groups. The patients underwent a clinical evaluation and laboratory, dosing sRAGE and glycated hemoglobin. sRAGE was measured in serum of patients by ELISA. Results: No statistically significant difference was found in plasma sRAGE levels between T2DM patients with and without microvascular complications, as well as no correlation between sRAGE and glycemic control. Conclusion: Further studies are needed to better elucidate the mechanisms involved in producing and regulating the concentration of soluble forms of RAGE and to clarify the cause-effect relationship between serum levels of sRAGE and the chronic complications of T2DM

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...

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

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