L'athérosclérose est une maladie chronique à évolution lente caractérisée par la formation de plaques d'athérome, consistant en l’accumulation de lipoprotéines de basse densité (LDL), de leucocytes, de cellules spumeuses, la migration des cellules musculaires lisses (CML) et l’altération des cellules endotheliales (ECs). Ces phénomènes conduisent à la formation d'un noyau nécrotique incluant des régions calcifiées. La genèse de l'athérosclérose et de l’instabilité de la plaque d’athérome sont le résultat d'une synergie entre inflammation et stress oxydant. Les données actuelles identifient plusieurs populations de macrophages dans la plaque d’athérome présentant différents phénotypes en lien avec l’inflammation (pro-inflammatoire: M1, anti-inflammatoire: M2) ou avec des modifications redox de l’environnement (Mox). Stress oxydant et inflammation sont liés et jouent un rôle important dans (i) la dysfonction endothéliale induisant une diminution de la biodisponibilité du monoxyde d’azote (NO), (ii) l'oxydation des LDL, (iii) le remodelage de la lésion (régulation de protéases et d’antiprotéases) et (iv) la prolifération des CML. Les CML sont le deuxième type cellulaire le plus abondant dans la lésion athérosclérotique après les macrophages, leur hyperprolifération est la conséquence d’une dédifférenciation cellulaire d’un phénotype contractile à sécrétoire, augmentant leur capacité proliférative et migratoire. Les donneurs de NO, comme les S-nitrosothiols, connus également pour protéger contre le stress oxydant grâce essentiellement à la S-nitrosation, peuvent contrer la carence en NO. Parmi eux, le S- nitrosoglutathion (GSNO), forme physiologique de stockage de NO dans les tissus, spécifiquement metabolisé par la gamma-glutamyl transférase (GGT) peut être envisagé. La corrélation entre l’augmentation des concentrations sériques de GGT et les facteurs de risque cardiovasculaire a récemment été démontrée. En particulier, seule la b-GGT s'accumule dans les plaques d'athérome, et concorde avec l’apparition d'autres marqueurs histologiques de vulnérabilité de la plaque. Étant donné que, les macrophages et les CML sont les principaux types cellulaires retrouvés dans les lésions athérosclérotiques et semblaient être colocalisés avec la GGT, l'attention de ce travail de thèse a été centrée sur la compréhension de la provenance de la GGT et son rôle dans le métabolisme du GSNO au sein de la plaque d’athérome. Une première partie de ce manuscrit vise à identifier l'origine de la GGT accumulée dans la plaque d’athérome, et à élucider entre le stimulus inflammatoire et oxydant, qui est responsable de l'accumulation de GGT dans la plaque d'athérome. La deuxième partie a été consacrée à la restauration de la biodisponibilité de NO dans les CML en condition de stress oxydant avec un intérêt particulier porté sur l'identification des protéines S-nitrosés / Atherosclerosis is a slowly progressing chronic disease characterized by the formation of atherosclerotic plaques consisting of accumulated low density lipoprotein (LDL), leukocytes, foam cells, migrated smooth muscle cells (SMCs) and altered endothelial cells (ECs), leading to the formation of necrotic cores with calcified regions. Atherosclerosis genesis and subsequent instability of atherosclerotic plaques result from a synergy between inflammation and oxidative stress. Current data identified several macrophage populations within the atherosclerotic plaque showing different inflammatory phenotypes (pro-inflammatory: M1, anti-inflammatory: M2) or functions in response to redox changes in the environment (Mox). The oxidative stress linked to inflammation plays an important role in (i) endothelial dysfunction, with reduced nitric oxide (NO) bioavailability, (ii) LDL oxidation, (iii) lesion remodeling (regulation of proteases and antiproteases) and (iv) SMCs proliferation. Indeed, SMCs are the second more abundant cell type, after macrophages, in the atherosclerotic lesion because their dedifferentiation from contractile to secreting phenotype increased their proliferation and migration capacity. NO donors, like S-nitrosothiols, also known to protect from oxidative stress by S-nitrosation, could counteract this NO deficiency. Among them, the S-nitrosoglutathione (GSNO), a physiological storage form of NO in tissues, specifically catabolized by the gamma-glutamyltransferase (GGT) is considered. Recently, it has been shown that the increased serum level of GGT is an independent risk factor for cardiovascular mortality related to atherosclerotic disease. In particular, only the big fraction (b-GGT) has been detected inside human atherosclerotic plaques associated to CD68+ macrophage-derived foam cells. As macrophages and SMCs are the main cell types found in atherosclerotic lesion and seemed to be colocalized with GGT, the attention of this thesis work was focused on the understanding of GGT provenance and its role in the GSNO metabolism within the atherosclerotic plaque. A first part of the thesis was to identify the origin of GGT accumulating inside atherosclerotic plaques, and to decipher between inflammation and oxidative stress stimuli, which one is responsible of GGT accumulation in atherosclerotic plaques. The second part was dedicated to the restoration of NO bioavailability within SMCs under oxidative stress with a focus on the identification of S-nitrosated proteins

<p>S-nitrosogluthatione is an endogenous substance, present at decreased levels in the lungs of CF patients and was recently found to induce mature CFTR in airway epithelial CF cell lines. We show that S-nitrosoglutathione in physiological concentrations increases the presence of ΔF508 CFTR in the cell membrane and induces cAMP dependent chloride transport in cystic fibrosis airway epithelial cells. The properties of S-nitrosoglutathione include other potential benefits for the CF patient and make this agent an interesting candidate for pharmacological treatment of CF that needs to be further evaluated.</p><p>Genistein was found to increase the chloride efflux in both normal and ΔF508 cells without stimulation of cAMP elevating agents and without prior treatment with phenylbutyrate. Genistein, in concentrations close to those that can be detected in plasma after a high soy diet, could induce chloride efflux in cells with the ΔF508 CFTR mutation and its possible use in the treatment of CF should therefore be further investigated.</p><p>Studies on nasal epithelial cells from CF patients showed cAMP dependent chloride efflux in some of the patients with severe genotypes. This may complicate <i>in vitro</i> evaluation of clinical treatment of these patients. The presence of cAMP dependent chloride transport did not necessarily lead to a milder phenotype. Other factors than CFTR may influence the clinical development of the disease.</p><p>Cystic fibrosis (CF) is the most common monogenetic disease among Caucasians. A defective cAMP regulated chloride channel (cystic fibrosis transmembrane conductance regulator, CFTR) in epithelial cells leads to viscous mucus, bacterial infections, inflammation and tissue damage in the lungs that cause death in 95% of the cystic fibrosis patients. There is no cure for the disease although existing treatment has dramatically prolonged the life expectancy. The aim of this thesis was to study pharmacological agents for their ability to restore the cellular deficiency in CF airway epithelial cells. X-ray microanalysis, MQAE fluorescence and immunocytochemistry were used to evaluate the effects.</p>

S-nitrosogluthatione is an endogenous substance, present at decreased levels in the lungs of CF patients and was recently found to induce mature CFTR in airway epithelial CF cell lines. We show that S-nitrosoglutathione in physiological concentrations increases the presence of ΔF508 CFTR in the cell membrane and induces cAMP dependent chloride transport in cystic fibrosis airway epithelial cells. The properties of S-nitrosoglutathione include other potential benefits for the CF patient and make this agent an interesting candidate for pharmacological treatment of CF that needs to be further evaluated. Genistein was found to increase the chloride efflux in both normal and ΔF508 cells without stimulation of cAMP elevating agents and without prior treatment with phenylbutyrate. Genistein, in concentrations close to those that can be detected in plasma after a high soy diet, could induce chloride efflux in cells with the ΔF508 CFTR mutation and its possible use in the treatment of CF should therefore be further investigated. Studies on nasal epithelial cells from CF patients showed cAMP dependent chloride efflux in some of the patients with severe genotypes. This may complicate in vitro evaluation of clinical treatment of these patients. The presence of cAMP dependent chloride transport did not necessarily lead to a milder phenotype. Other factors than CFTR may influence the clinical development of the disease. Cystic fibrosis (CF) is the most common monogenetic disease among Caucasians. A defective cAMP regulated chloride channel (cystic fibrosis transmembrane conductance regulator, CFTR) in epithelial cells leads to viscous mucus, bacterial infections, inflammation and tissue damage in the lungs that cause death in 95% of the cystic fibrosis patients. There is no cure for the disease although existing treatment has dramatically prolonged the life expectancy. The aim of this thesis was to study pharmacological agents for their ability to restore the cellular deficiency in CF airway epithelial cells. X-ray microanalysis, MQAE fluorescence and immunocytochemistry were used to evaluate the effects.

Indiana University-Purdue University Indianapolis (IUPUI) / S-nitrosoglutathione reductase (GSNOR) also known as glutathione- dependent formaldehyde dehydrogenase (FDH), is a zinc-dependent dehydrogenase. GSNOR oxidizes long chain alcohols to an aldehyde with the help of a molecule of NAD+. GSNOR was initially identified as FDH because of its role in the formaldehyde detoxification pathway. The only S-nitrosothiol (SNO) substrate recognized by GSNOR is GSNO. A transnitrosation reaction transfers NO from nitrosylated proteins or S-nitrosothiols (RSNO) to glutathione to form S-nitrosoglutathione. This GSNO is finally converted to glutathione disulfide (GSSG) by a two step mechanism. Cellular GSNO is a nitric oxide reservoir that can either transfer to or remove from the proteins a NO group. Reduction of GSNO by GSNOR depletes this reservoir and therefore indirectly regulates protein nitrosylation. GSNOR inhibitors which can increase the basal GSNO levels will be another potential therapy. Several GSNOR inhibitors were identified in our laboratory and the aim of this study was to understand their cellular effects. One of the experiments studied the effect of the compound on protein-SNO. The role of nitric oxide in regulation of NF-κB pathway is reviewed by Bove and van der Vliet. We focused on identification of nitrosylated proteins using protein specific antibodies. We identified nitrosylation of IKKβ. So the question raised was whether nitrosylation of IKKβ affects its activity. IKKβ is responsible for phosphorylation of IκBα and phosphorylation of IκBα results in its degradation and activation of NF-κB pathway. Therefore, we studied the phosphorylation of IκBα in the presence of inhibitor C3. Results showed a dose-dependent decrease of pIκB. So the next question was whether the phosphorylation of IKKβ was affected by nitrosylation. We did not detect any change in pIKKβ with different concentrations of C3. The decreased degradation of IκBα caused by C3 translated into decreased NF-κB activity as seen by a dose-dependent decrease in amounts of ICAM-1 with increasing C3 concentration. This data supports the premise that the activity of transcription factor NF-κB is suppressed by inhibiting GSNOR with compound C3

Submitted by Jos? Henrique Henrique (jose.neves@ufvjm.edu.br) on 2017-09-12T20:11:13Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) regina_inez_souza.pdf: 2322505 bytes, checksum: fb22e286692f3f78b323a1aa5767c86b (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2017-09-18T13:03:41Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) regina_inez_souza.pdf: 2322505 bytes, checksum: fb22e286692f3f78b323a1aa5767c86b (MD5) / Made available in DSpace on 2017-09-18T13:03:41Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) regina_inez_souza.pdf: 2322505 bytes, checksum: fb22e286692f3f78b323a1aa5767c86b (MD5) Previous issue date: 2017 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / Na ind?stria farmac?utica, os pol?meros s?o bastante utilizados em sistemas para libera??o controlada de f?rmacos, por serem capazes de liber?-los efetivamente no local alvo, aumentando os benef?cios terap?uticos e minimizando os efeitos colaterais. Entre os pol?meros utilizados para essa finalidade, tem-se o poli(3-hidroxibutirato) (PHB) pertencente a classe dos poli(hidroxialcanoatos), sendo um pol?mero de origem microbiana, que destacase por ser biorreabsorv?vel e biocompat?vel. Quando utilizado em misturas f?sicas com outros pol?meros, as denominadas blendas, modifica-se propriedades f?sicas, mec?nicas e biol?gicas do PHB para aumentar sua aplicabilidade. Nesse sentido, o propilenoglicol (PPG) foi utilizado para prepara??o de blendas PHBPPG (90/10). O objetivo do trabalho foi preparar e estudar filmes polim?ricos constitu?dos por PHB e PHBPPG (90/10) contendo 1% e 5% de Snitrosoglutationa (GSNO), um doador de ?xido n?trico (NO) bem como sintetizar o f?rmaco e avaliar sua estabilidade ap?s armazenamento a 23?C, 4?C e -18?C durante 90 dias por an?lise t?rmica e por espectroscopia na regi?o do infravermelho. Os filmes obtidos contendo GSNO foram caracterizados por an?lise t?rmica, espectroscopia no infravermelho e avaliou-se o perfil de libera??o in vitro dos mesmos. A citotoxicidade dos extratos das matrizes constitu?das somente por PHB e PHBPPG (90/10) foi avaliada sobre c?lulas mononucleares do sangue perif?rico (PBMC) e leuc?citos totais pelo m?todo de exclus?o com azul de Tripan e sobre hem?cias pela avalia??o da atividade hemol?tica. Os resultados da an?lise t?rmica e infravermelho mostraram que a melhor forma de armazenamento de GSNO ? em 4?C e -18?C. As matrizes polim?ricas de PHB e da blenda PHBPPG (90/10) contendo 1% e 5% de GSNO, avaliadas por an?lise t?rmica foram capazes de liberar NO e apresentaram-se mais est?veis termicamente quando comparados aos filmes de PHB e da blenda sem o f?rmaco. O perfil de libera??o dos filmes de PHB e da blenda PHBPPG (90/10) mostrou que h? uma libera??o r?pida inicial de GSNO nas 24 horas iniciais seguido por um padr?o de libera??o controlada no per?odo de 1 a 8 dias para os filmes contendo 5% de GSNO, sendo o filme da blenda PHBPPG (90/10) contendo 5% de f?rmaco, o que apresentou maior porcentagem de libera??o no per?odo avaliado. Extratos provenientes dos filmes de PHB e da blenda PHBPPG (90/10) n?o apresentaram car?ter citot?xico por n?o induzirem hem?lise das hem?cias e n?o reduzirem a viabilidade celular de PBMCs e leuc?citos totais. As blendas desenvolvidas neste trabalho possuem a capacidade de libera??o controlada e in situ de ?xido n?trico, e possuem aplica??o potencial, por exemplo, no revestimento de stents coronarianos para preven??o da restenose p?s-angioplastia. / Disserta??o (Mestrado) ? Programa de P?s-gradua??o em Ci?ncias Farmac?uticas, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2017. / Polymers are widely used for controlled drug release systems improving the therapeutic benefits and minimizing side effects by the pharmaceutical industry due their capability of releasing it effectively at the target site. Among the polymers used for this purpose there is poly(3-hydroxybutyrate, PHB), a polymer from microbial origin of the poly(hydroxyalkanoate) class that is both bioresorbable and biocompatible. When used as a physical blends with other polymers several properties of PHB (physical, mechanical and biological) could be modified to increase their applicability. To produce blends with PHB the propyleneglycol (PPG) was used to prepare PHBPPG blends (90/10). The aim of this work was to prepare and study polymeric films composed by PHB and PHBPPG (90/10) containing 1% and 5% S-nitrosoglutathione (GSNO), a nitric oxide (NO) donor. The GSNO was synthesized and had its stability evaluated after storage at 23 ? C, 4 ? C and -18 ? C for 90 days by thermal analysis and infrared spectroscopy. The polymer materials containing GSNO were characterized by thermal analysis, infrared spectroscopy and also their drug release capability was evaluated. The cytotoxicity of matrices constituted by PHB and PHBPPG (90/10) was evaluated on peripheral blood mononuclear cells (PBMC) and total leukocytes by the exclusion method with Tripan blue and on red blood cells by the evaluation of hemolytic activity. The thermal and infrared analysis showed that the best form of GSNO storage is at 4 ? C and -18 ? C. Polymeric matrices of PHB and PHBPPG (90/10) containing 1% and 5% of GSNO, were able to release NO and were more thermally stable when compared to PHB and blends without the drug. The release profile of the PHB and PHBPPG blends (90/10) showed an initial fast release of GSNO at the initial 24 hours followed by a controlled release pattern in the period of 1 to 8 days for materials containing 5% of GSNO. The PHBPPG (90/10) blends containing 5% of drug presented the highest percentage of release in the evaluated period. The PHB and PHBPPG (90/10) blends did not present cytotoxic effects. The studied materials did not induced hemolysis of the red blood cells and did not reduced the cellular viability of PBMCs and total leukocytes. The developed blends are capable to exert the in situ controlled-release of nitric oxide and can potentially be used for example to coat coronary stents and thus help to prevent post-angioplasty restenosis.

Les S-nitrosothiols (RSNO) comme le S-nitrosoglutathion (GSNO) sont des donneurs de monoxyde d’azote (NO) prometteurs pour le traitement des maladies cardiovasculaires. Cependant, ce sont des candidats médicaments peu stables. Précédemment, des nanoparticules chargées en GSNO (GSNO-NP) ont été incluses dans une matrice d’alginate/chitosan. Les particules composites ainsi produites avaient une bonne encapsulation et une libération prolongée de GSNO. De plus, leur administration orale à des rats produisait un stock de NO au niveau de la paroi de l’aorte. Elles avaient cependant plusieurs limitations : préparation et caractérisation longues, manque de stabilité et de reproductibilité. Ce travail avait donc trois objectifs : (1) déterminer le mécanisme d’absorption intestinale des RSNO non formulés ; (2) évaluer la capacité des RSNO non formulés à créer un stock vasculaire de NO ; 3) optimiser la formulation de GSNO. Nous avons montré, dans un modèle in vitro de barrière intestinale, que la perméabilité intestinale de GSNO, S-nitroso-N-acétylcystéine (NACNO) et S-nitroso-N-acetylpénicillamine (SNAP) se fait par un mécanisme passif, principalement par voie transcellulaire (également paracellulaire pour SNAP), avec une perméabilité moyenne. Après avoir traversé la barrière intestinale, les RSNO atteindront les vaisseaux sanguins. Pour comparer leur capacité à former un stock vasculaire de NO dans des aortes (avec endothélium intact ou retiré), nous avons quantifié le stock, vérifié sa biodisponibilité pour la vasorelaxation et évalué son impact sur une vasoconstriction induite par la phénylephrine (PHE). L’incubation des aortes avec les RSNO augmente le stock basal de NO par un facteur trois à cinq. Ce stock est mobilisable pour induire la vasorelaxation et efficace pour diminuer la réactivité vasculaire à la PHE (NACNO>GSNO = SNAP), seulement dans les aortes dont l’endothélium a été retiré. Comme la perméabilité intestinale des RSNO est moyenne, l’intégration du GSNO dans une formulation appropriée est nécessaire. Vu l’impossibilité de résoudre les problèmes liés aux particules composites, le protocole de production des GSNO-NP a été modifié pour produire des microparticules (deux types selon l’état liquide ou solide de GSNO dans la phase interne de l’émulsion). Les deux types de microparticules avaient une libération de GSNO ralentie par rapport aux GSNO-NP. Les nano- comme les micro-particules ont pu être stabilisées par lyophilisation, et amélioraient la perméabilité intestinale de GSNO (jusqu’à une forte perméabilité avec les microparticules). Par conséquent, une administration orale de nano/microparticules chargées en GSNO/RSNO pourrait représenter une nouvelle approche thérapeutique pour les maladies cardiovasculaires. / S-nitrosothiols (RSNOs) such as S-nitrosoglutathione (GSNO) are promising nitric oxide (NO) donors for cardiovascular diseases treatment. However, they are poorly stable drug candidates. In previous studies, GSNO-loaded nanoparticles (GSNO-NP) were embedded into an alginate/chitosan matrix. Resulting nanocomposite particles showed high encapsulation and sustained release of GSNO, and led to the formation of a NO store in the wall of aorta after a single oral administration to rats. However, these nanocomposite particles have several limitations such as time-consuming preparation, lack of both stability and reproducibility. This thesis work aimed at: 1) Elucidate the mechanism of free RSNOs intestinal absorption; 2) Evaluate ability of free RSNOs to form a vascular NO store; 3) Optimize the GSNO formulation. In this study, we showed that the intestinal permeability (in vitro model of intestinal barrier) of GSNO, S-nitroso-N-acetylcysteine (NACNO) and S-nitroso-N-acetylpenicillamine (SNAP) was a passive diffusion, following the transcellular pathway (and also the paracellular way for SNAP) and belonging to the medium permeability class. After crossing the intestinal barrier, RSNOs will reach the vasculature. In order to compare the ability of free RSNOs to form a vascular store of NO either in endothelium-intact or endothelium-removed aortae, we quantified the store, verified its bioavailability for vasorelaxation and evaluated its impact on phenylephrine (PHE)-induced vasoconstriction. Incubation with RSNOs increased the basal NO store three to five times. This store is still bioavailable to induce vasorelaxation and efficient to induce vascular hyporeactivity to PHE (NACNO> GSNO = SNAP) only in endothelium-removed aortae. As intestinal permeability of RSNOs was in the medium class, the integration of GSNO into an appropriate delivery system is essential. Limitations of previously developed nanocomposites particles were impossible to bypass so the production process of GSNO-NP was modified (liquid or solid GSNO in the internal phase of the emulsion) to produce microparticles. Both kinds of microparticles exhibited a slower release of GSNO than GSNO-NP. Nano-and micro-particles were stable after lyophilization and presented an enhancement of GSNO intestinal permeability (up to high permeability class for microparticles). Thus, oral administration of GSNO/RSNO loaded nano/micro particles seems to be a promising avenue for the treatment of cardiovascular diseases.

No description available.