No description available.

Beim ischämischen Schlaganfall führt ein thrombotischer Verschluss von gehirnversorgenden Arterien zu einer akuten Durchblutungsstörung, mit der Folge von neurologischen Defiziten. Primäres Therapieziel ist es, diese Blutgerinnsel aufzulösen, um die Sauerstoffversorgung des Gehirns wiederherzustellen und den ischämischen Hirnschaden zu begrenzen. Dazu stehen die intravenösen Thrombolyse mit rt-PA (rekombinanter Gewebe-Plasminogen-Aktivator) sowie die endovaskuläre mechanische Thrombektomie zur Verfügung. Häufig kann ein Schlaganfall, trotz erfolgreicher Rekanalisation der Gefäße, zu einer weiteren Größenzunahme des Infarktes und neurologischen Defiziten bei den Patienten führen. Diese Größenzunahme beruht zum einen auf einem sich entwickelnden Hirnödem und zum anderen auf entzündlichen Prozessen. Zahlreiche Hinweise deuten darauf hin, dass der Schlaganfall ein Zusammenspiel aus thrombotischen und entzündlichen Ereignissen ist, ein Phänomen, das als Thromboinflammation bezeichnet wird. Aufgrund der begrenzten Behandlungsmöglichkeiten ist die Entwicklung neuer Therapieansätze für den ischämischen Schlaganfall besonders wichtig. Agaphelin und Ixolaris sind Proteine aus den Speicheldrüsen von Hämatophagen, für welche in früheren Studien eine starke antithrombotische Wirkung bei gleichzeitig geringem Blutungsrisiko nachgewiesen wurde. Diese möglichen antithrombotischen Effekte wurden in dieser Studie im Hinblick auf ihre Wirksamkeit und Sicherheit im Mausmodell der zerebralen Ischämie untersucht. Die Behandlung der Mäuse mit Agaphelin 1 Stunde nach transienter Okklusion der Arteria cerebri media (tMCAO) führte zu kleineren Schlaganfallvolumina und geringeren neurologischen Defiziten an Tag 1 nach dem Schlaganfall. Die Mortalität der Mäuse war bis Tag 7 deutlich gesunken. Aus klinischer Sicht ist ebenfalls relevant, dass der starke antithrombotische Effekt von Agaphelin im Mausmodell nicht mit einem erhöhten Risiko für intrazerebrale Blutungen einherging. Diesem protektiven Effekt von Agaphelin lagen eine verminderte intrazerebrale Thrombusbildung, eine abgeschwächte Entzündungsantwort und eine Stabilisierung der Blut-Hirn-Schranke sowie eine Reduzierung der Apoptose zugrunde. Nach der Gabe von Ixolaris 1 Stunde nach tMCAO waren zwar signifikant geringere Infarktgrößen messbar, diese führten allerdings nicht zu einer Verbesserung der neurologischen Defizite. Zudem verursachte die Gabe von Ixolaris schon 24 Stunden nach tMCAO erhebliche intrazerebrale Blutungen und auch die Mortalität der Mäuse war zu diesem Zeitpunkt bereits erhöht. Aufgrund dieser massiven Nebenwirkungen scheint Ixolaris kein geeigneter Kandidat für eine humane Anwendung zu sein. Bei Agaphelin hingegen könnte es sich um einen vielversprechenden Kandidaten für die Behandlung des ischämischen Schlaganfalls handeln. Vor einer möglichen Testung von Agaphelin in klinischen Studien, sind weitere translationale Untersuchungen notwendig, um ein noch präziseres Verständnis für die Wirksamkeit und Sicherheit von Agaphelin zu gewinnen. Insgesamt stellt die Hemmung thromboinflammatorischer Prozesse, ohne eine Erhöhung der Blutungskomplikationen, eine vielversprechende Option zur Behandlung des ischämischen Schlaganfalls dar. / Thrombotic occlusion of cerebral vessels is an important process in pathogenesis of ischemic stroke resulting in lack of blood supply of the brain and neurological deficits. In order to restore the oxygenation of the brain and to limit brain injury, recanalization of the occluded vessels is the therapeutic main goal of stroke treatment. So far, the only proven pharmacological intervention for thrombolysis is the recombinant tissue-type plasminogen activator. For recanalization of larger arteries endovascular thrombectomy was established as a mechanic intervention. Nevertheless, despite successful recanalization ischemic brain damage and neurological deficits evolve. Increased size of infarct lesions develop due to brain edema and inflammatory processes. Moreover, there is evidence that inflammation and thrombosis are linked, which has led to the concept of thromboinflammation. Due to the limited treatment strategies in stroke management, the development of new therapeutic approaches for ischemic stroke is particularly important. Recent studies have shown that the hematophagous salivary gland proteins Agaphelin and Ixolaris exhibit multiple antithrombotic effects without promoting a risk of bleeding. To investigate the potentially safe antithrombotic effects, Agaphelin and Ixolaris were tested in a mouse model of transient middle cerebral artery occlusion (tMCAO). Treatment of mice with Agaphelin 1 hour after tMCAO resulted in smaller infarct volumes and an improved neurological function on day one after stroke. Up to seven days after stroke the mortality rate was significantly reduced. This protective effect was due to reduced local thrombus formation, a reduced inflammatory response and less severe blood-brain-barrier damage as well as reduced apoptosis. Moreover, it is important to mention, that the strong protective effect of Agaphelin was not linked to an increased risk of intracerebral bleeding. Treatment of mice with Ixolaris one hour after tMCAO leads to significantly smaller infarct sizes. However, neurologic deficits did not improve after treatment with Ixolaris. Furthermore, risk of intracerebral bleeding and mortality rates were significantly increased 24 hours after treatment with Ixolaris. Due to these severe side effects, Ixolaris does not seem to be an appropriate candidate for human therapy. Nevertheless, Agaphelin appears to be a promising component for ischemic stroke treatment, but further translational studies should be performed before testing Agaphelin in clinical stroke trials. Overall, the inhibition of thromboinflammatory effects without increased bleeding reflects a promising option for successful ischemic stroke treatment.

Aortic valve replacement (AVR) is the only life-saving treatment for patients with severe symptomatic aortic stenosis. Bioprosthetic valves are used in 90% of AVRs because they do not require lifelong anticoagulation. The major limitation of bioprosthetic valves is their limited durability compared to mechanical valves. In addition, bioprosthetic valves still carry a 2-3% risk of symptomatic valve thrombosis, stroke, and thromboembolism in the first 30 days after implantation, and a 1% annual risk thereafter. The risk of subclinical valve thrombosis is around 10% at 30 days and 25% at 1 year, and prevention of subclinical valve thrombosis is hypothesized to reduce the risk of clinical thrombotic events and perhaps even improve valve durability, although high-quality evidence is lacking. This doctoral thesis comprises 7 chapters of varied methodology that summarize the evidence behind current recommendations for antithrombotic therapy after bioprosthetic AVR, identify evidence gaps, and present the design a randomized trial that aims to address some of these evidence gaps. Chapter 1 introduces each included study with a brief summary. Chapter 2 is a narrative review summarizing guideline recommendation for antithrombotic therapy after bioprosthetic AVR and the evidence upon which they are based. Chapter 3 is an observational study describing antithrombotic prescribing practices in the VISION Cardiac Surgery cohort study. Chapter 4 is a systematic review and network meta-analysis of randomized studies of antithrombotic therapy after transcatheter aortic valve replacement. Chapter 5 is a systematic review and meta-analysis of randomized and observational studies of subclinical valve thrombosis. Chapter 6 presents the design and rationale of a feasibility trial of direct oral anticoagulants versus vitamin K antagonists in patients with a new surgical bioprosthetic AVR and atrial fibrillation. Chapter 7 discusses the implications, limitations, and future avenues of the research presented in this doctoral thesis. / Thesis / Doctor of Philosophy (PhD) / More than 10,000 Canadians require aortic valve replacement each year. Bioprosthetic valves (made out of cow or pig tissue) are often preferred over mechanical valves (made out of metal) because the risk of blood clots forming on the valve or causing a stroke is lower. The disadvantage of bioprosthetic valves is that they can wear out and require re-replacement. The reason why bioprosthetic valves wear out is uncertain, but it may be related to small blood clots on the valve that are only detectable on a CT scan. This doctoral thesis explores the use of blood thinners for patients with bioprosthetic aortic valve replacement. Ideally, blood thinners would be able to prevent blood clots and stroke, and to improve the durability of bioprosthetic valves, without causing too much bleeding. The thesis reviews the available evidence, identifies unanswered questions, and ends with a proposal for a study to generate new data.

ADAMTS13 is a metalloprotease that regulates the length, and thus, the platelet-capturing capacity of von Willebrand factor. The regulation of ADAMTS13 activity remains poorly understood. Numerous circulating proteases cleave ADAMTS13 in vitro, impairing its activity, but the physiological significance of this mechanism remains unknown. Two commonly cleaved regions within ADAMTS13 were identified and mutants were developed: two with one of each region mutated (T4L and T8L mutants), one with both regions mutated (T4L/T8L or “double” mutant), and one with an additional elastase site mutated (T4L/T8L + I380G). This work characterizes the mutants’ resistance to proteolysis and compares the activity of the double mutant to wild-type ADAMTS13 (WT). Each mutant and WT was incubated with purified coagulation and neutrophil proteases, activated neutrophils, or added to plasma before initiating coagulation with or without tissue plasminogen activator. Cleavage patterns were visualized with western blot. FRETS-VWF73 and microfluidic flow assays were used to compare WT and mutant activity. Coagulation proteases cleave both predicted sites within WT, and the double mutant exhibits near complete resistance to cleavage over 3 hours. Resistance to degradation by neutrophil proteases is prolonged in the double mutant, but additional cleavage sites are present. Elastase cleavage is prevented in the T4L/T8L + I380G mutant. In plasma, WT is degraded upon initiating coagulation and subsequent fibrinolysis, which is prevented in the double mutant. WT is also degraded in the presence of activated neutrophils, and the double and T4L/T8L + I380G mutants exhibit improved but incomplete resistance. Finally, the mutants exhibit similar activity to WT using FRETS-VWF73 and the microfluidic assay. This work validates the location of two protease-sensitive regions within ADAMTS13 and confirms the resistance of the double mutant to coagulation proteases in vitro. Future work will complete the activity analysis, and compare the mutants’ therapeutic efficacy to WT in vivo. / Thesis / Master of Science (MSc) / Current drugs used to dissolve blood clots can cause major bleeding. Therefore, safer treatments need to be developed. An important step in the clotting pathway is platelet accumulation in the injured vessel. Platelets stick to string-like protein, von Willebrand Factor (VWF), and ADAMTS13 is a protein that regulates this by cutting VWF strings. ADAMTS13 shows promise as a treatment for clots without causing bleeding, but it is unclear how its activity is controlled. ADAMTS13 can be degraded by other proteins, however the importance of this process in the body is unknown. This work characterizes a degradation-resistant ADAMTS13 mutant, which may be used to study whether ADAMTS13 degradation reduces its therapeutic effectiveness. The mutant has normal VWF-cutting activity, is resistant to degradation by clotting proteins, and is partially resistant to proteins released by neutrophils, an important immune cell in clotting. Future studies will investigate its effectiveness at treating clots in animals.

No description available.

Coronary artery disease and chronic pain are serious health issues that cause severe discomfort and suffering in society today. Antithrombotic agents and highly potent analgesics play a critical role in improving the recovery process for patients being treated for these diseases. This thesis focuses on the design and study of pellet-based drug dosage forms which allow diffusion-controlled delivery of drugs with the aim of achieving optimal therapeutic outcomes. A wet slurry process was used to mix the drug and the polymer and/or clay precursor excipients into a paste. The pellets were then shaped via ionotropic gelation (alginate hydrogel beads/pellets), extrusion/spheronization (halloysite clay pellets) or geopolymerization. The decrease in the drug diffusion rate in the alginate beads was affected by the drug's molecular size and charge and the characteristics (such as concentration and chemical structure) of the surrounding alginate gel. The halloysite clay pellets provided sustained release of the highly potent drug fentanyl at both gastric pH 1 and intestinal pH 6.8. As expected, crushing the pellets reduced the diffusion barrier, resulting in more rapid release (dose dumping). The use of mechanically strong geopolymer gels was investigated as a potential means of preventing dose dumping as a result of crushing of the dosage form. Variations in the synthesis composition resulted in drastic changes in the microstructure morphology, the porosity, the mechanical stability and the drug release rate. Pore network modeling and finite element simulations were employed to theoretically evaluate the effects of porosity and drug solubility in the geopolymer structure on the drug release process. Fitting the model parameters to experimental data showed that increased average pore connectivity, a greater pore size distribution, and increased drug solubility in the pellet resulted in an increased drug release rate. Furthermore, incorporation of pH-sensitive organic polymers in the geopolymer structure reduced the high drug release rate from the pellets at gastric pH. These results indicate that geopolymers have potential for use in pellet form; both the release rate of the drug and the mechanical stability of the pellets can be optimized to prevent dose dumping.

QUINDERE, Ana Luiza Gomes. Caracterização estrutural e atividade anti-inflamatória em modelo de aterosclerose em camundongos de uma galactana sulfatada da alga vermelha Acanthophora muscoides. 2015. 152 f. Tese (Doutorado em Bioquímica)-Universidade Federal do Ceará, Fortaleza, 2015. / Submitted by Vitor Campos (vitband@gmail.com) on 2016-09-01T22:42:17Z No. of bitstreams: 1 2015_tese_algquindere.pdf: 1798137 bytes, checksum: c21776a201b808de159ff9bc73b36644 (MD5) / Approved for entry into archive by Jairo Viana (jairo@ufc.br) on 2016-09-02T21:05:07Z (GMT) No. of bitstreams: 1 2015_tese_algquindere.pdf: 1798137 bytes, checksum: c21776a201b808de159ff9bc73b36644 (MD5) / Made available in DSpace on 2016-09-02T21:05:07Z (GMT). No. of bitstreams: 1 2015_tese_algquindere.pdf: 1798137 bytes, checksum: c21776a201b808de159ff9bc73b36644 (MD5) Previous issue date: 2015 / Sulfated galactans from red marine algae are polysaccharides with heterogeneous structures that have presented a variety of potentially therapeutic biological effects including anticoagulant, anti-thrombotic and anti-inflammatory, however, their potential activity as anti-inflammatory agent in the treatment of atherosclerosis, a chronic inflammatory disease that culminates with thromboembolic disorders, has not been previously studied. Furthermore, experimental data from animal models and clinical studies support connections between the hemostasis and inflammation in atherogenesis. These interfaces among inflammation and thrombogenesis have been suggested as targets for pharmacological intervention to reduce disease progression. Herein, we determined the chemical structure of a novel sulfated galactan obtained from the marine alga Acanthophora muscoides (fraction AmII) and analysed its effect on a mice model of atherosclerosis in 10-week aged apolipoprotein E deficient (ApoE−/−) mice under high-cholesterol diet for additional 4 or 11 weeks. Fraction AmII (10 mg/kg) or Vehicle were subcutaneously injected from week 2 until 4 of the diet or from week 6 until week 11 of the diet. In vitro assays of macrophage chemotaxis were also performed. The structure of the complex sulfated galactan was characterized by solution nuclear magnetic resonance and its molecular mass was determined by gel permeation chromatography and polyacrylamide gel electrophoresis. The sulfated galactan from A. muscoides presents a molecular mass of ~ 20kDa and an alternating 4-linked α-galactose and 3-linked β-galactose, substituted with sulfate esters and methyl ethers along with the occurrence of 3,6-anhydro-α-galactoses. In the 4 weeks diet model, treatment with fraction AmII did not alter the atherosclerotic plaque size, and other intraplaque features of vulnerability (such as lipid, neutrophil, macrophage, MMP-9 and collagen contents). In the 11 weeks diet model, treatment with fraction AmII reduced intraplaque macrophage and tissue factor (TF) content as compared to Vehicle-treated animals. Intraplaque TF co-localized and positively correlated with macrophage rich-areas. No changes on atherosclerotic plaque size, and other intraplaque features of vulnerability, such as lipid, neutrophil, MMP-9 and collagen contents, were observed. Moreover, mRNA expression of MMPs, chemokines and genetic markers of Th1/2/reg/17 lymphocyte polarization within mouse aortic arches and spleens was not affected by AmII treatment. In vitro, treatment with AmII dose-dependently reduced macrophage chemotaxis without affecting TF production. Overall, the chronic AmII treatment was well tolerated. In conclusion, our results indicate that AmII treatment reduced intraplaque macrophage content, by impacting on cell recruitment, and, concomitantly, intraplaque TF content of potential macrophage origin in atherosclerotic mice. / As galactanas sulfatadas obtidas de algas marinhas vermelhas são polissacarídeos de estruturas heterogêneas que têm apresentado uma variedade de efeitos biológicos, potencialmente terapêuticos, incluindo anticoagulante, antitrombótico e anti-inflamatório. No entanto, a sua atividade potencial como agente anti-inflamatório para o tratamento de aterosclerose, uma doença inflamatória crônica que culmina com distúrbios tromboembólicos, não foi previamente estudada. Além disso, dados experimentais de modelos animais e estudos clínicos suportam conexões entre a hemostasia e inflamação na aterogênese. Estas interfaces entre inflamação e trombogênese têm sido sugeridas como alvos para intervenção farmacológica visando reduzir a progressão da doença. No presente trabalho, determinou-se a estrutura química de uma nova galactana sulfatada obtida da alga marinha Acanthophora muscoides (fração AmII) e analisou-se seu efeito sobre um modelo de aterosclerose em camundongos deficientes em apolipoproteína E (ApoE−/−) de 10 semanas submetidos a dieta de alto teor de colesterol durante mais 4 ou 11 semanas. A fração AmII (10 mg/kg) ou veículo (salina) foram injetados por via subcutânea durante a segunda até a quarta semana de dieta ou a partir da sexta até a décima primeira semana de dieta. Ensaios in vitro de quimiotaxia de macrófagos também foram realizados. A estrutura da galactana sulfatada complexa foi caracterizada por ressonância magnética nuclear em solução e a sua massa molecular foi determinada por cromatografia de permeação em gel e eletroforese em gel de poliacrilamida. A fração AmII apresentou uma massa molecular de ~ 20 kDa e uma alternância de α-galactose 4 ligada e β-galactose 3-ligada, substituído com ésteres de sulfato e éteres de metil, juntamente com a ocorrência de unidades de 3,6-anidro-α-galactoses. No modelo de dieta de 4 semanas, o tratamento com a fração AmII não alteraou o tamanho da placa aterosclerótica e demais características de vulnerabilidade intraplaca, tais como lípido, neutrófilos, macrófagos, metaloprotease de matriz (MMP)-9 e conteúdo de colágeno. No modelo de dieta de 11 semanas, o tratamento com a fração AmII reduziu os conteúdos de macrófago intraplaca e de fator tecidual (FT), em comparação com animais tratados com veículo. O FT intraplaca co-localizou e positivamente correlacionou com áreas ricos em macrófagos. Não foram observadas alterações no tamanho da placa aterosclerótica e nas outras características de vulnerabilidade intraplaca, tais como conteúdos de lipídios, neutrófilos, MMP-9 e do colágeno. Além disso, a expressão de mRNA de MMPs, quimiocinas e marcadores genéticos de polarização de linfócitos Th1/2/reg/17 nos arcos aórticos e nos baços dos camundongos não foi alterada pelo tratamento AmII. In vitro, o tratamento com AmII reduziu de forma dose-dependente a quimiotaxia de macrófagos sem afetar a produção de FT. No geral, o tratamento crônico com AmII foi bem tolerado. Em conclusão, nossos resultados indicam que o tratamento com AmII em camundongos ateroscleróticos reduziu o conteúdo de macrófagos intraplaca, agindo sobre o recrutamento celular, e, concomitantemente, o conteúdo de FT intraplaca de originado potencialmente de macrófagos.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Sulfated galactans from red marine algae are polysaccharides with heterogeneous structures that have presented a variety of potentially therapeutic biological effects including anticoagulant, anti-thrombotic and anti-inflammatory, however, their potential activity as anti-inflammatory agent in the treatment of atherosclerosis, a chronic inflammatory disease that culminates with thromboembolic disorders, has not been previously studied. Furthermore, experimental data from animal models and clinical studies support connections between the hemostasis and inflammation in atherogenesis. These interfaces among inflammation and thrombogenesis have been suggested as targets for pharmacological intervention to reduce disease progression. Herein, we determined the chemical structure of a novel sulfated galactan obtained from the marine alga Acanthophora muscoides (fraction AmII) and analysed its effect on a mice model of atherosclerosis in 10-week aged apolipoprotein E deficient (ApoE−/−) mice under high-cholesterol diet for additional 4 or 11 weeks. Fraction AmII (10 mg/kg) or Vehicle were subcutaneously injected from week 2 until 4 of the diet or from week 6 until week 11 of the diet. In vitro assays of macrophage chemotaxis were also performed. The structure of the complex sulfated galactan was characterized by solution nuclear magnetic resonance and its molecular mass was determined by gel permeation chromatography and polyacrylamide gel electrophoresis. The sulfated galactan from A. muscoides presents a molecular mass of ~ 20kDa and an alternating 4-linked α-galactose and 3-linked β-galactose, substituted with sulfate esters and methyl ethers along with the occurrence of 3,6-anhydro-α-galactoses. In the 4 weeks diet model, treatment with fraction AmII did not alter the atherosclerotic plaque size, and other intraplaque features of vulnerability (such as lipid, neutrophil, macrophage, MMP-9 and collagen contents). In the 11 weeks diet model, treatment with fraction AmII reduced intraplaque macrophage and tissue factor (TF) content as compared to Vehicle-treated animals. Intraplaque TF co-localized and positively correlated with macrophage rich-areas. No changes on atherosclerotic plaque size, and other intraplaque features of vulnerability, such as lipid, neutrophil, MMP-9 and collagen contents, were observed. Moreover, mRNA expression of MMPs, chemokines and genetic markers of Th1/2/reg/17 lymphocyte polarization within mouse aortic arches and spleens was not affected by AmII treatment. In vitro, treatment with AmII dose-dependently reduced macrophage chemotaxis without affecting TF production. Overall, the chronic AmII treatment was well tolerated. In conclusion, our results indicate that AmII treatment reduced intraplaque macrophage content, by impacting on cell recruitment, and, concomitantly, intraplaque TF content of potential macrophage origin in atherosclerotic mice. / As galactanas sulfatadas obtidas de algas marinhas vermelhas sÃo polissacarÃdeos de estruturas heterogÃneas que tÃm apresentado uma variedade de efeitos biolÃgicos, potencialmente terapÃuticos, incluindo anticoagulante, antitrombÃtico e anti-inflamatÃrio. No entanto, a sua atividade potencial como agente anti-inflamatÃrio para o tratamento de aterosclerose, uma doenÃa inflamatÃria crÃnica que culmina com distÃrbios tromboembÃlicos, nÃo foi previamente estudada. AlÃm disso, dados experimentais de modelos animais e estudos clÃnicos suportam conexÃes entre a hemostasia e inflamaÃÃo na aterogÃnese. Estas interfaces entre inflamaÃÃo e trombogÃnese tÃm sido sugeridas como alvos para intervenÃÃo farmacolÃgica visando reduzir a progressÃo da doenÃa. No presente trabalho, determinou-se a estrutura quÃmica de uma nova galactana sulfatada obtida da alga marinha Acanthophora muscoides (fraÃÃo AmII) e analisou-se seu efeito sobre um modelo de aterosclerose em camundongos deficientes em apolipoproteÃna E (ApoE−/−) de 10 semanas submetidos a dieta de alto teor de colesterol durante mais 4 ou 11 semanas. A fraÃÃo AmII (10 mg/kg) ou veÃculo (salina) foram injetados por via subcutÃnea durante a segunda atà a quarta semana de dieta ou a partir da sexta atà a dÃcima primeira semana de dieta. Ensaios in vitro de quimiotaxia de macrÃfagos tambÃm foram realizados. A estrutura da galactana sulfatada complexa foi caracterizada por ressonÃncia magnÃtica nuclear em soluÃÃo e a sua massa molecular foi determinada por cromatografia de permeaÃÃo em gel e eletroforese em gel de poliacrilamida. A fraÃÃo AmII apresentou uma massa molecular de ~ 20 kDa e uma alternÃncia de α-galactose 4 ligada e β-galactose 3-ligada, substituÃdo com Ãsteres de sulfato e Ãteres de metil, juntamente com a ocorrÃncia de unidades de 3,6-anidro-α-galactoses. No modelo de dieta de 4 semanas, o tratamento com a fraÃÃo AmII nÃo alteraou o tamanho da placa aterosclerÃtica e demais caracterÃsticas de vulnerabilidade intraplaca, tais como lÃpido, neutrÃfilos, macrÃfagos, metaloprotease de matriz (MMP)-9 e conteÃdo de colÃgeno. No modelo de dieta de 11 semanas, o tratamento com a fraÃÃo AmII reduziu os conteÃdos de macrÃfago intraplaca e de fator tecidual (FT), em comparaÃÃo com animais tratados com veÃculo. O FT intraplaca co-localizou e positivamente correlacionou com Ãreas ricos em macrÃfagos. NÃo foram observadas alteraÃÃes no tamanho da placa aterosclerÃtica e nas outras caracterÃsticas de vulnerabilidade intraplaca, tais como conteÃdos de lipÃdios, neutrÃfilos, MMP-9 e do colÃgeno. AlÃm disso, a expressÃo de mRNA de MMPs, quimiocinas e marcadores genÃticos de polarizaÃÃo de linfÃcitos Th1/2/reg/17 nos arcos aÃrticos e nos baÃos dos camundongos nÃo foi alterada pelo tratamento AmII. In vitro, o tratamento com AmII reduziu de forma dose-dependente a quimiotaxia de macrÃfagos sem afetar a produÃÃo de FT. No geral, o tratamento crÃnico com AmII foi bem tolerado. Em conclusÃo, nossos resultados indicam que o tratamento com AmII em camundongos aterosclerÃticos reduziu o conteÃdo de macrÃfagos intraplaca, agindo sobre o recrutamento celular, e, concomitantemente, o conteÃdo de FT intraplaca de originado potencialmente de macrÃfagos.

L’augmentation de la prévalence des hémorragies cérébrales (HC) sous traitement antithrombotique constitue un problème de Santé Publique à cause du mauvais pronostic vital et fonctionnel et du risque compétitif de récidive hémorragique ou ischémique chez les survivants, qui pose le problème d’une prévention secondaire adaptée._x000D_L’objectif de la thèse était d’étudier l’impact des traitements antithrombotiques sur le pronostic des HC. Le terme d’HC, entendu au sens large, regroupe les HC spontanées et les microhémorragies cérébrales.La première partie avait comme objectif de comparer les caractéristiques des HC survenant avec ou sans traitement par antagonistes de la vitamine K (AVK), stratifiant l’analyse selon la localisation de l’HC. L’analyse a porté sur 545 patients de la cohorte Lilloise (PITCH), parmi lesquels 83 (15%) étaient sous AVK. La prise d’un traitement par AVK n’influençait pas la localisation de l’HC, mais était prédictive d’HC plus volumineuses (25 ml vs 12 ; p=0.002) chez les victimes d’HC non lobaire, alors que aucune différence existait en cas d’HC lobaire (26 ml vs 30; p=0.507). Ces résultats suggèrent que l’impact des AVK diffère en fonction de la localisation de l’HC et de la vasculopathie sous-jacente.La deuxième partie avait comme objectif d’analyser l’impact du traitement antithrombotique sur l’apparition de nouvelles microhémorragies cérébrales stratifiant l’analyse selon la localisation de l’HC. L’étude a porté sur 168 survivants à 6 mois d’une HC (cohorte PITCH) ayant bénéficié d’une IRM cérébrale 1.5T lors de l’HC et durant le suivi. Lors de l’HC, 89 (53%) patients présentaient des microhémorragies, et 80 (48%) ont développé des microhémorragies durant le suivi. La présence sur la première IRM de microhémorragies (aOR 2,3; IC 95%1,2-4,3), leur position mixte, lobaire et profonde (aOR 3.7; IC95% 1,7-8,3), et la présence de séquelles de macrohémorragies (aOR 6,8; IC95% 2,8-16,7), étaient associées à l’apparition de microhémorragies. Chez les patients avec HC non lobaire, l’apparition de microhémorragies était associée à l’utilisation d’un traitement antithrombotique durant le suivi (aOR 2,9 ; IC95% 1,1-7,3) et avec l’apparition de lacunes (aOR: 2,9; 95%CI 1,0-7,8). Chez les patients avec HC lobaire, l’apparition de microhémorragies était associée à l’apparition de macrohémorragie (aOR 9.8 ; IC95% 1,1-88,8). Ces résultats suggèrent que l’impact des traitements antithrombotiques diffère en fonction de la vasculopathie sous-jacente.La troisième partie avait comme objectif de : (i) comparer les proportions de survivants d’une HC ayant une indication formelle au traitement antithrombotique chez lesquels ce traitement était repris à la sorite ; (ii) identifier les caractéristiques associées à la reprise du traitement antithrombotique, au sein de 5 cohortes européennes (Lille, n=542; Utrecht, n=389 ; Amsterdam, n= 403 ; Londres, n=667 ; Lothian, n=137). Un traitement antithrombotique était recommencé chez 96 (20%) des 469 survivants ayant une indication formelle, mais en proportions différentes dans les 5 cohortes (Lille 18%, Utrecht 45%, Amsterdam 30% ; Londres 11% ; Lothian 15%; p<0.001). Nous n’avons retrouvé aucun autre facteur prédictif de reprise du traitement antithrombotique en dehors de la cohorte d’origine. Ces résultats montrent que la décision de reprise du traitement antithrombotique ne repose pas sur des critères reproductibles, et soulignent la nécessité de réaliser des essais randomisés.La quatrième partie, actuellement en cours, prévoit d’évaluer chez ces mêmes patients (survivants d’une HC ayant une indication formelle au traitement antithrombotique) le risque de récidive hémorragique ou ischémique en fonction de la reprise ou de l’arrêt du traitement antithrombotique. Il s’agit d’une étude observationnelle portant sur 274 patients de 4 cohortes Européennes, avec un suivi d’environ 2.5 ans. L’analyse statistique est en cours. / The proportion of patients who are taking antithrombotic drugs at the time they suffer a spontaneous intracerebral hemorrhage (ICH) is increasing over time, and this is a major issue in public health because of the poor prognosis of patients. Also, the decision to restart or not antithrombotic drugs in survivors is still a clinical dilemma. _x000D_The aim of this work was to evaluate the impact of antithrombotic drugs on ICH prognosis. The term ICH regroups (i) spontaneous ICH and (ii) cerebral microbleeds (CMBs).As a first step, we compared baseline characteristics of 545 consecutive patients with ICH included in the PITCH cohort (Lille, France), receiving or not Vitamin K Antagonists (VKAs) at the time of ICH, stratifying the analysis according to the ICH location (lobar vs non lobar). VKAs-ICH accounted for 83 patients (15%). The use of VKAs did not influence ICH location, but influenced ICH volume: in non lobar ICH, VKAs use was associated with larger ICH volumes (25 ml versus 12 ml, p=0.002). In lobar ICH, no difference was observed (26ml versus 30ml; p=0.507). The different impact of VKAs on ICH volumes according to location suggests a different susceptibility of the underlying vasculopathies to VKAs.As a second step, we aimed to evaluate the impact of antithrombotic drugs on the incidence of CMBs in 168 ICH survivors from the PITCH cohort who underwent 1.5T Magnetic resonance imaging (MRI) at ICH onset and during follow-up, stratifying the analysis according to the index ICH location. At the time of ICH, 89 (53%) patients had CMBs at ICH onset, and 80 (48%) showed new CMBs during follow-up. Predictors of incident CMBs were the presence on the first MRI of: at least 1 CMB (aOR 2.3; 95% CI: 1.2-4.3), old macro-hemorrhage (aOR 6.8; 95%CI 2.8-16.7), and CMBs in mixed location (aOR 3.7; 95%CI 1.7-8.3). In non lobar ICH, incident CMBs were associated with incident lacunes (aOR: 2.9; 95%CI 1.0-7.8) and with the use of antiplatelet agents (aOR 2.9; 95%CI 1.1-7.3). In lobar ICH, incident CMBs were associated with incident macro-hemorrhage (aOR 9.8; 95%CI 1.1-88.8). These results suggest that the impact of antithrombotic drugs differs according to the index ICH location, and therefore according to the underlying vasculpathy.The third step consisted in comparing the characteristics and proportions of patients taking antithrombotic drugs at ICH discharge in five European cohorts (Lille, France, n=542; Utrecht, The Netherlands, n=389; Amsterdam, The Netherlands, n=403; multicenter UK cohort, n=667; Lothian, Scotland, n=137). We then identified characteristics associated with restarting. Antithrombotic drugs were restarted in 96 (20%) of the 469 survivors who had an indication for antithrombotic drugs (secondary prevention or atrial fibrillation), but in different proportions according to the cohort of origin (Lille 18%, Utrecht 45%, Amsterdam 30%, CROMIS-2 ICH 11%, Lothian 15%; p<0.001). We did not find other consistent associations with restarting antithrombotic drugs. The variation in clinical practice in restarting antithrombotic drugs after ICH supports the need for randomized controlled trials.In the fourth step, we aim to analyse the risk of ICH recurrences or ischemic events in the same population of patients (ICH survivors who suffered from ICH while on antithrombotic drugs because of secondary prevention or atrial fibrillation) according to the antithrombotic drug status during follow-up. We included 274 patients from 4 European cohorts, with a median follow-up of 2.5 years. Statistical analysis is ongoing.

Submitted by Rosivalda Pereira (mrs.pereira@ufma.br) on 2017-05-22T16:51:30Z No. of bitstreams: 1 SamiraAbdala.pdf: 3051742 bytes, checksum: 816c5c802400bcf5a75deb6a99de0617 (MD5) / Made available in DSpace on 2017-05-22T16:51:30Z (GMT). No. of bitstreams: 1 SamiraAbdala.pdf: 3051742 bytes, checksum: 816c5c802400bcf5a75deb6a99de0617 (MD5) Previous issue date: 2017-04-07 / Fundação de Amparo à Pesquisa e ao Desenvolvimento Científico e Tecnológico do Maranhão (FAPEMA) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPQ) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Platelets, the blood cells involved in maintaining hemostasis, play a key role in the development of acute ischemic coronary, cerebrovascular events and are critically involved in the thrombosis process. In response to vascular injury, changes in blood flow or chemical stimuli, platelets trigger three functional mechanisms: adhesion, activation and aggregation. After platelet capture, a rapid stabilization of adhesion is required for thrombus formation to occur. Platelet activation results from conformational changes dependent of the protein disulfide isomerase (PDI), so that it has recently been proposed as a molecular target in platelet antiaggregant activity. The use of plant species rich in phenolic compounds as a source of bioactive substances is a promising strategy for the development of new therapeutic alternatives for thromboembolic diseases. Previously, we have shown that Syzygium cumini (L.) Skeels leaf contains multiple polyphenols, which support its use for antiplatelet purposes. Therefore, this study sought to evaluate the effects of polyphenol-rich extract (PESc) from S. cumini leaf on platelet activation and aggregation, as well as on PDI reductase activity. Platelet-rich plasma from healthy volunteers (n=5) was incubated with PESc (10-1000 μg/mL), for 25 min, before activation with ADP, thrombin or PMA. To analyze PESc effect on integrin αIIbβ3 activation, flow citometry protocols were conducted in washed platelets pre-treated with PESc (10-1000μg/mL) and activated with thrombin before tagging with PAC–1 antibody. Finally, PESc (0.1-100 μg/mL) effects on PDI reductase activity were assessed in absence or presence of polyphenolic standards gallic acid, myricetin and quercetin. PESc dose-dependently inhibited platelet aggregation despite the agonist used, even though lower agonist concentration potentiated PESc inhibitory effects to a maximal 77% inhibition at 2.5 μM ADP. Similarly, PESc dose-dependently reduced the proportion of activated αIIbβ3 molecules per platelet up to one third of control at 1000 μg/mL. These effects correlated with the strong inhibitory action of PESc on PDI activity, an effect synergically augmented in presence of standards. Therefore, our data show that PESc reduces platelet aggregation and activation, probably through PDI inhibition, strengthening its prominent antiplatelet activity. / As plaquetas, células sanguíneas envolvidas na manutenção da hemostase, exercem uma função essencial no desenvolvimento de eventos isquêmicos agudos coronarianos, cerebrovasculares e estão criticamente envolvidas no processo de formação da trombose. Em resposta à lesão vascular, às alterações no fluxo sanguíneo ou a estímulos químicos, as plaquetas desencadeiam três mecanismos funcionais: adesão, ativação e agregação. Após a captura da plaqueta, uma rápida estabilização da adesão é necessária para que ocorra a formação do trombo. A ativação plaquetária resulta de alterações conformacionais dependentes da proteína dissulfeto isomerase (PDI), de tal modo, que recentemente foi proposto o seu uso como alvo molecular na atividade antiagregante plaquetária. O uso de espécies vegetais ricas em compostos fenólicos como fonte de substâncias bioativas apresenta-se como uma estratégia promissora para o desenvolvimento de novas alternativas terapêuticas das doenças tromboembólicas. Anteriormente, temos mostrado que as folhas de Syzygium cumini (L.) Skeels contém múltiplos polifenóis, que apoiam a sua utilização para fins antiplaquetários. Portanto, este estudo procurou avaliar os efeitos do extrato rico em polifenóis (ERP) da folha de S. cumini sobre a ativação e agregação plaquetária, bem como, sobre a atividade redutase da PDI. Para tanto, plasma rico em plaquetas de voluntários saudáveis foi incubado com ERP (10 - 1000μg/mL), durante 25 min, antes da ativação com ADP, trombina ou PMA. Para analisar o efeito de ERP sobre a ativação da integrina αIIbβ3, os protocolos de citometria de fluxo foram conduzidos em plaquetas lavadas pré-tratadas com ERP (10 -1000 μg/mL) e ativadas com trombina antes da marcação com anticorpo PAC-1. Finalmente, os efeitos de ERP (0,1-100 μg/mL) na atividade redutase da PDI foram avaliados na ausência ou presença de padrões polifenólicos de ácido gálico, miricetina e quercetina. ERP inibiu a agregação plaquetária dependente da dose apesar do agonista utilizado, embora uma menor concentração de agonistas potencializasse os efeitos inibitórios de ERP até uma inibição máxima de 77% a 2,5 μM de ADP. De modo semelhante, o ERP reduziu a dose proporcionalmente a proporção de moléculas de αIIbβ3 ativadas por plaquetas até um terço do controle a 1000μg/mL. Estes efeitos correlacionaram-se com a forte ação inibitória de ERP na atividade da PDI, um efeito sinergicamente aumentado na presença de padrões. Portanto, nossos dados mostram que o ERP reduz a agregação e ativação plaquetária, provavelmente através da inibição da PDI, fortalecendo sua proeminente atividade antiplaquetária.