A deficiência da desidrogenase de acilas-CoA de cadeia média (MCAD) é um defeito da oxidação de ácidos graxos (DOAG) bioquimicamente caracterizada pelo acúmulo de acilcarnitinas de cadeia média (MCAC) em tecidos e líquidos biológicos de indivíduos afetados. Clinicamente os pacientes apresentam sintomas neurológicos como letargia e coma, geralmente desencadeados por episódios de descompensação metabólica. Outros distúrbios importantes entre os DOAG são as deficiências da proteína trifuncional mitocondrial (MTPD) e da desidrogenase de 3-hidroxiacilas-CoA de cadeia longa (LCHADD) que são caracterizadas pelo acúmulo de ácidos graxos de cadeia longa e seus derivados 3- hidroxilados (LCHFA). Geralmente, os pacientes afetados apresentam disfunção cardíaca e hepática podendo também apresentar sinais de comprometimento neurológico. Até o momento, a hipoglicemia e a toxicidade dos ácidos graxos acumulados têm sido relacionados com a fisiopatologia dessas doenças, embora os mecanismos responsáveis pelo dano tecidual não são bem conhecidos. Assim, foram investigados os efeitos dos MCAC e LCHFA sobre importantes parâmetros da função redox e homeostase energética mitocondrial em cérebro e coração de ratos, bem como a produção de superóxido e morte celular em culturas de fibroblastos da pele de pacientes afetados por essas doenças. Primeiramente, observamos que as MCAC induzem dano oxidativo lipídico e proteico, além de reduzir as defesas antioxidantes não enzimáticas em cérebro de ratos, provavelmente através da indução de radicais hidroxil e peroxil. Além disso, evidenciamos aumento dos níveis de superóxido e de morte celular em fibroblastos de pacientes afetados pela MTPD em condições padrão de cultivo indicando uma exposição crônica a níveis mais elevados de superóxido e uma vulnerabilidade à morte celular. Fibroblastos de pacientes afetados pelas MTPD e MCADD também apresentaram aumento dos níveis de superóxido quando cultivadas em condições de estresse metabólico, contudo não houve aumento de morte celular. Finalmente, a morte celular foi mais pronunciada em fibroblastos afetados pela MTPD em comparação com pacientes afetados pela MCADD, além de apresentar uma forte correlação com a produção de superóxido, sugerindo que esses eventos são provavelmente associados. Por outro lado, observamos que LCHFA prejudicam a homeostase energética em mitocôndrias de coração devido a um efeito desacoplador da fosforilação oxidativa evidenciado pelo aumento do estado 4 da respiração e diminuição da razão de controle respiratório, pela redução do potencial de membrana, do conteúdo de NAD(P)H e da produção de H2O2. O ácido 3- hidroxitetradecanóico (3 HTA) também induziu inchaço mitocondrial provavelmente como consequência da abertura do poro de transição de permeabilidade mitocondrial (mPTP) uma vez que a ciclosporina A (CsA), um inibidor clássico da abertura do poro, impediu esse efeito em mitocôndrias cardíacas carregadas com Ca2+. LCHFA também dissipadaram o potencial de membrana, diminuíram o coneúdo de NAD(P)H e de ATP na presença ou ausência de Ca2+ em mitocondriais de córtex cerebral. Além disso, na presença de Ca2+, 3 HTA induziu inchamento mitocondrial e liberação de citocromo c, que ativa rotas apoptóticas. 3 HTA também afetou a homeostase de Ca2+ evidenciado por uma reduzida capacidade de retenção de Ca2+ pela mitocôndria e induziu a produção de H2O2 na presença de Ca2+. Essas alterações foram prevenidas pelo vermelho de rutênio (RR), um bloqueador da captação mitocondrial de Ca2+, e por CsA mais ADP, inibidores da abertura do mPTP, o que implica o seu envolvimento nesses efeitos. Em contraste, LCHFA não causaram dano oxidativo nem alteraram as defesas antioxidantes em coração de ratos jovens. Em conjunto, nós demonstramos que MCAC e LCHFA que se acumulam em pacientes afetados pelas deficiências da MCAD e MTP/LCHAD, respectivamente, são potencialmente tóxicos para as funções mitocondriais essenciais em cérebro e coração de ratos. Além disso, demonstramos que fibroblastos de pacientes com MTPD cultivados em condições padrão estão sob estresse oxidativo, que é acentuado quando os fibroblastos são submetidos a condições de estresse metabólico. Presume-se que esses mecanismos fisiopatológicos podem ser responsáveis, pelo menos em parte, pelo dano tecidual característico desses pacientes. / The medium chain acyl-CoA dehydrogenase (MCAD) deficiency is a fatty acid oxidation disorder (FAOD) biochemically characterized by accumulation of medium-chain acylcarnitines (MCAC) in tissues and biological fluids of affected individuals. The clinical presentation includes neurological symptoms as lethargy and coma, generally followed by episodes of metabolic decompensation. Other important FAOD are the mitochondrial trifunctional protein (MTPD) and the long-chain 3- hydroxyacyl-CoA dehydrogenase (LCHADD) deficiencies that are characterized by accumulation of long-chain fatty acids and their 3-hydroxylated (LCHFA) derivatives. Usually, affected patients present cardiac and hepatic dysfunction and may present signs of neurological impairment. So far, hypoglycemia and the toxicity of accumulating fatty acids have been related with the pathophysiology in these disorders, although the mechanisms responsible for tissue damage are poorly known. Thus, we investigated the effects of MCAC and LCHFA on important parameters of mitochondrial redox and energetic homeostasis in brain and heart of rats, as well as superoxide production and cell death in skin fibroblasts from patients affected by these diseases. First, we observed that MCAC induced lipid and protein oxidative damage, and reduced the antioxidant non-enzymatic defenses in rat brain, probably through induction of hydroxyl and peroxyl radicals. Furthermore, we evidenced increased superoxide levels and cell death in skin fibroblasts from MTP deficient patients under standard growing conditions indicating a chronic exposure to higher superoxide levels and a vulnerability to cell death. In addition, cells from MCADD and MTPD patients cultured under metabolic stress conditions presented increased levels of superoxide, although cell death was not increased. Finally, cell death was more pronounced in fibroblasts from MTP compared to MCAD deficient patients, and presented a strong correlation with superoxide production, suggesting that these events are probably associated. On the other hand, we observed that LCHFA provoked an impairment of heart mitochondrial energetic homeostasis, by behaving as uncouplers of oxidative phosphorylation, evidenced by increase of state 4 respiration and decrease of the respiratory control ratio, by reducing the membrane potential, the NAD(P)H content and H2O2 production. 3-Hydroxytetradecanoic acid (3 HTA) also induced mitochondrial swelling probably as a consequence of the mitochondrial permeability transition pore (mPTP) opening once cyclosporin A (CsA), a classical inhibitor, prevented this effect in heart mitochondria loaded with Ca2+. LCHFA also dissipated membrane potential, diminished NAD(P)H content and decreased ATP content in the presence or absence of Ca2+ in cerebral cortex mitochondrial preparations. Moreover, 3 HTA induced mitochondrial swelling and cytochrome c release in the presence of Ca2+, which activates apoptotic cascades. 3 HTA also affected Ca2+ homeostasis evidenced by a diminished mitochondrial Ca2+ retention capacity and induced hydrogen peroxide production in the presence of Ca2+. These alterations where prevented by ruthenium red (RR), a blocker of mitochondrial Ca2+ uptake, and by CsA plus ADP, inhibitors of the mPTP, implying its involvement in these effects. In contrast, LCHFA did not cause oxidative damage nor altered the antioxidant defenses in heart of young rats. Taken together, we demonstrated that MCAC and LCHFA found accumulated in patients affected by MCAD and MTP/LCHAD deficiencies, respectively, are potentially toxic to essential mitochondrial functions in rat brain and heart. We also found that fibroblasts from MTP deficient patients cultured under basal growing conditions are under oxidative stress that is accentuated when cultured metabolic stress conditions are used. It is presumed that these pathomechanisms may be responsible, at least in part, for the tissue damage characteristic of these patients.

A deficiência da desidrogenase de acilas-CoA de cadeia média (MCAD) é um defeito da oxidação de ácidos graxos (DOAG) bioquimicamente caracterizada pelo acúmulo de acilcarnitinas de cadeia média (MCAC) em tecidos e líquidos biológicos de indivíduos afetados. Clinicamente os pacientes apresentam sintomas neurológicos como letargia e coma, geralmente desencadeados por episódios de descompensação metabólica. Outros distúrbios importantes entre os DOAG são as deficiências da proteína trifuncional mitocondrial (MTPD) e da desidrogenase de 3-hidroxiacilas-CoA de cadeia longa (LCHADD) que são caracterizadas pelo acúmulo de ácidos graxos de cadeia longa e seus derivados 3- hidroxilados (LCHFA). Geralmente, os pacientes afetados apresentam disfunção cardíaca e hepática podendo também apresentar sinais de comprometimento neurológico. Até o momento, a hipoglicemia e a toxicidade dos ácidos graxos acumulados têm sido relacionados com a fisiopatologia dessas doenças, embora os mecanismos responsáveis pelo dano tecidual não são bem conhecidos. Assim, foram investigados os efeitos dos MCAC e LCHFA sobre importantes parâmetros da função redox e homeostase energética mitocondrial em cérebro e coração de ratos, bem como a produção de superóxido e morte celular em culturas de fibroblastos da pele de pacientes afetados por essas doenças. Primeiramente, observamos que as MCAC induzem dano oxidativo lipídico e proteico, além de reduzir as defesas antioxidantes não enzimáticas em cérebro de ratos, provavelmente através da indução de radicais hidroxil e peroxil. Além disso, evidenciamos aumento dos níveis de superóxido e de morte celular em fibroblastos de pacientes afetados pela MTPD em condições padrão de cultivo indicando uma exposição crônica a níveis mais elevados de superóxido e uma vulnerabilidade à morte celular. Fibroblastos de pacientes afetados pelas MTPD e MCADD também apresentaram aumento dos níveis de superóxido quando cultivadas em condições de estresse metabólico, contudo não houve aumento de morte celular. Finalmente, a morte celular foi mais pronunciada em fibroblastos afetados pela MTPD em comparação com pacientes afetados pela MCADD, além de apresentar uma forte correlação com a produção de superóxido, sugerindo que esses eventos são provavelmente associados. Por outro lado, observamos que LCHFA prejudicam a homeostase energética em mitocôndrias de coração devido a um efeito desacoplador da fosforilação oxidativa evidenciado pelo aumento do estado 4 da respiração e diminuição da razão de controle respiratório, pela redução do potencial de membrana, do conteúdo de NAD(P)H e da produção de H2O2. O ácido 3- hidroxitetradecanóico (3 HTA) também induziu inchaço mitocondrial provavelmente como consequência da abertura do poro de transição de permeabilidade mitocondrial (mPTP) uma vez que a ciclosporina A (CsA), um inibidor clássico da abertura do poro, impediu esse efeito em mitocôndrias cardíacas carregadas com Ca2+. LCHFA também dissipadaram o potencial de membrana, diminuíram o coneúdo de NAD(P)H e de ATP na presença ou ausência de Ca2+ em mitocondriais de córtex cerebral. Além disso, na presença de Ca2+, 3 HTA induziu inchamento mitocondrial e liberação de citocromo c, que ativa rotas apoptóticas. 3 HTA também afetou a homeostase de Ca2+ evidenciado por uma reduzida capacidade de retenção de Ca2+ pela mitocôndria e induziu a produção de H2O2 na presença de Ca2+. Essas alterações foram prevenidas pelo vermelho de rutênio (RR), um bloqueador da captação mitocondrial de Ca2+, e por CsA mais ADP, inibidores da abertura do mPTP, o que implica o seu envolvimento nesses efeitos. Em contraste, LCHFA não causaram dano oxidativo nem alteraram as defesas antioxidantes em coração de ratos jovens. Em conjunto, nós demonstramos que MCAC e LCHFA que se acumulam em pacientes afetados pelas deficiências da MCAD e MTP/LCHAD, respectivamente, são potencialmente tóxicos para as funções mitocondriais essenciais em cérebro e coração de ratos. Além disso, demonstramos que fibroblastos de pacientes com MTPD cultivados em condições padrão estão sob estresse oxidativo, que é acentuado quando os fibroblastos são submetidos a condições de estresse metabólico. Presume-se que esses mecanismos fisiopatológicos podem ser responsáveis, pelo menos em parte, pelo dano tecidual característico desses pacientes. / The medium chain acyl-CoA dehydrogenase (MCAD) deficiency is a fatty acid oxidation disorder (FAOD) biochemically characterized by accumulation of medium-chain acylcarnitines (MCAC) in tissues and biological fluids of affected individuals. The clinical presentation includes neurological symptoms as lethargy and coma, generally followed by episodes of metabolic decompensation. Other important FAOD are the mitochondrial trifunctional protein (MTPD) and the long-chain 3- hydroxyacyl-CoA dehydrogenase (LCHADD) deficiencies that are characterized by accumulation of long-chain fatty acids and their 3-hydroxylated (LCHFA) derivatives. Usually, affected patients present cardiac and hepatic dysfunction and may present signs of neurological impairment. So far, hypoglycemia and the toxicity of accumulating fatty acids have been related with the pathophysiology in these disorders, although the mechanisms responsible for tissue damage are poorly known. Thus, we investigated the effects of MCAC and LCHFA on important parameters of mitochondrial redox and energetic homeostasis in brain and heart of rats, as well as superoxide production and cell death in skin fibroblasts from patients affected by these diseases. First, we observed that MCAC induced lipid and protein oxidative damage, and reduced the antioxidant non-enzymatic defenses in rat brain, probably through induction of hydroxyl and peroxyl radicals. Furthermore, we evidenced increased superoxide levels and cell death in skin fibroblasts from MTP deficient patients under standard growing conditions indicating a chronic exposure to higher superoxide levels and a vulnerability to cell death. In addition, cells from MCADD and MTPD patients cultured under metabolic stress conditions presented increased levels of superoxide, although cell death was not increased. Finally, cell death was more pronounced in fibroblasts from MTP compared to MCAD deficient patients, and presented a strong correlation with superoxide production, suggesting that these events are probably associated. On the other hand, we observed that LCHFA provoked an impairment of heart mitochondrial energetic homeostasis, by behaving as uncouplers of oxidative phosphorylation, evidenced by increase of state 4 respiration and decrease of the respiratory control ratio, by reducing the membrane potential, the NAD(P)H content and H2O2 production. 3-Hydroxytetradecanoic acid (3 HTA) also induced mitochondrial swelling probably as a consequence of the mitochondrial permeability transition pore (mPTP) opening once cyclosporin A (CsA), a classical inhibitor, prevented this effect in heart mitochondria loaded with Ca2+. LCHFA also dissipated membrane potential, diminished NAD(P)H content and decreased ATP content in the presence or absence of Ca2+ in cerebral cortex mitochondrial preparations. Moreover, 3 HTA induced mitochondrial swelling and cytochrome c release in the presence of Ca2+, which activates apoptotic cascades. 3 HTA also affected Ca2+ homeostasis evidenced by a diminished mitochondrial Ca2+ retention capacity and induced hydrogen peroxide production in the presence of Ca2+. These alterations where prevented by ruthenium red (RR), a blocker of mitochondrial Ca2+ uptake, and by CsA plus ADP, inhibitors of the mPTP, implying its involvement in these effects. In contrast, LCHFA did not cause oxidative damage nor altered the antioxidant defenses in heart of young rats. Taken together, we demonstrated that MCAC and LCHFA found accumulated in patients affected by MCAD and MTP/LCHAD deficiencies, respectively, are potentially toxic to essential mitochondrial functions in rat brain and heart. We also found that fibroblasts from MTP deficient patients cultured under basal growing conditions are under oxidative stress that is accentuated when cultured metabolic stress conditions are used. It is presumed that these pathomechanisms may be responsible, at least in part, for the tissue damage characteristic of these patients.

Orientador: Otavio Rizzi Coelho / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-11T03:33:19Z (GMT). No. of bitstreams: 1 Marsaro_EliandraAparecida_D.pdf: 2012256 bytes, checksum: 4f444be0b390a7068513571b25f2a026 (MD5) Previous issue date: 2008 / Resumo: Introdução: Estudos têm sugerido que as estatinas aumentam a sobrevida em pacientes portadores de insuficiência cardíaca com função sistólica preservada, benefício provavelmente atribuído aos seus efeitos pleiotrópicos em co-morbidades como doença arterial coronariana e hipertrofia de ventrículo esquerdo. Objetivos: avaliar se o uso de sinvastatina em dose máxima por um período de 6 meses pode alterar parâmetros ecoDopplercardiográficos de função diastólica em pacientes hipertensos com disfunção diastólica isolada. Pacientes e Métodos: Este é um estudo aleatorizado duplo-cego placebo controlado. Foram estudados 30 indivíduos hipertensos normocolesterolêmicos com idade = 50 anos sem evidência de hipertrofia de ventrículo esquerdo ou isquemia miocárdica investigada por ecocardiografia de estresse. Os pacientes foram aleatorizados para placebo ou sinvastatina (80mg) e reavaliados após 6 meses. Resultados: Os grupos Sinvastatina e Placebo apresentavam características semelhantes no início do estudo. Após 6 meses de acompanhamento, 67% dos pacientes do grupo Sinvastatina apresentaram normalização da função diastólica contra 33% do grupo Placebo (p<0.05). Todos os indivíduos tratados com sinvastatina que não apresentaram normalização da função diastólica tiveram aumentos da pressão arterial sistólica e diastólica após os 6 meses (p<0.05 para ambas). Conclusão: O tratamento de hipertensos com sinvastatina 80mg/d por 6 meses foi associado a uma maior taxa de normalização da função diastólica quando comparado a placebo. O aumento da pressão arterial observado em alguns indivíduos aparentemente aboliu esse benefício / Abstract: Background: Therapy with statins was suggested to improve survival on heart failure with preserved systolic function. This benefit was attributed probably to their pleiotropic effects on co-morbidities like coronary heart disease or left ventricular hypertrophy. Aims: evaluate the impact of the treatment with simvastatin on echocardiografic parameters of diastolic function in hypertensive patients without coronary heart disease or left ventricular hypertrophy. Patients and methods: This is a randomized, placebo-controlled, double-blind study. We studied 30 normocholesterolemic hypertensive patients = 50 years without evidence of left ventricular hypertrophy or coronary artery disease assessed under rest and stress echocardiography. The patients have been randomized for placebo or simvastatin (80mg) and reevaluated after 6 months. Results: The baseline characteristics were similar between groups. After six months of treatment, 67% of the patients of simvastatin group presented normalization of diastolic function against 33% in the placebo group (p<0.05). All subjects in simvastatin group who not presented normalization of the diastolic function have increased on systolic and diastolic blood pressure after 6 months (p<0.05 for both). Conclusion: This study demonstrates that a 6 months therapy with simvastatin 80mg/d in asymptomatics hypertensive subjects with diastolic dysfunction had a higher rate of normalization of diastolic function than placebo. The increased of blood pressure observed in some subjects in the simvastatin group appearentelly abolished this benefit / Doutorado / Clinica Medica / Mestre em Clinica Medica

As dislipidemias são importantes fatores de risco para desenvolvimento de Doenças Cardiovasculares (DCV) comprovado através de grandes estudos observacionais. Estudos demonstraram que a prescrição regular de hipolipemiantes (estatinas e fibratos) pode reduzir a ocorrência de eventos cardiovasculares e diminuir a mortalidade. Objetivou-se realizar uma análise econômica e a influência de atorvastatina, sinvastatina, bezafibrato e ciprofibrato sobre a morbimortalidade cardiovascular em indivíduos que fizeram uso destes medicamentos em 2007 dispensados no Programa de Medicamentos Excepcionais do Ministério da Saúde distribuídos pela Farmácia Ambulatorial do Hospital das Clínicas da FMRP-USP. Trata-se de um estudo observacional, descritivo e de caráter transversal. A casuística foi composta por 332 (31,11%) indivíduos sorteados aleatoriamente dentre 1067 pacientes (erro padrão de 5%), de ambos os sexos, encaminhados pelo Sistema Único de Saúde (SUS) e consultórios particulares. Os indivíduos selecionados foram submetidos a uma entrevista e seus prontuários médicos analisados. Dos 310 pacientes entrevistados, 157 (51%) eram do sexo masculino. A faixa etária variou de 15 a 63 anos (X= 62,0 ± 12,23). Constatou-se 5 óbitos em 2007, sendo 100% do sexo masculino, com idade variando de 57 a 74 anos (X= 68,2 ± 6,95). 227 (73,22%) pacientes fizeram uso de estatinas, 54 (17,42%) de fibratos e 31 (10%) controles (sem uso de medicamentos). De 246 (79,35%) indivíduos analisados, a média do índice de massa corpórea (IMC) foi >28,7 Kg/m2; 121 (39%) pacientes fizeram uso de sinvastatina, 104 (34%) atorvastatina, 25 (8%) ciprofibrato, 29 (9%) bezafibrato. O perfil lipídico apresentou-se mais elevado no grupo atorvastatina e bezafibrato. Em relação aos eventos e/ou procedimentos, houve um total de 253. 132 (52,17%) pacientes apresentaram aterosclerose documentada, 60 (23,71%), angina pectoris, 28 (11,47%) insuficiência cardíaca, 6 (2,44%) infarto agudo do miocárdio, 6 (2,44%) aneurisma arterial e 4 (1,62%) acidente vascular encefálico. Quanto aos procedimentos, constatou-se a realização de 11 cateterismos cardíacos e 7 angioplastias. Quanto à análise econômica, o tratamento do grupo atorvastatina apresentou o maior custo (R$994,69 paciente/ano), já no grupo da sinvastatina (R$337,61 paciente/ano) houve maiores gastos com exames laboratoriais e complementares. Entre o grupo dos fibratos não houve diferenças importantes com relação ao custo do tratamento. Conclui-se que entre os indivíduos estudados, prevaleceu a população idosa, maior número de óbitos no sexo masculino; houve a prevalência de sobrepeso/obesidade (IMC > 25 kg/m2); aterosclerose documentada e angina pectoris foram os eventos cardiovasculares predominantes e o cateterismo cardíaco o procedimento mais realizado. O tratamento com atorvastatina foi o mais oneroso, entretanto, seus pacientes apresentaram menor ocorrência de eventos e procedimentos cardiovasculares, além do menor custo com exames laboratoriais. / Dyslipidemias are a major risk factor for the development of cardiovascular diseases. Several studies have shown that regular prescribing of lipid-lowering drugs (statins and fibrates) can reduce cardiovascular events and decrease overall mortality. The objectives of this study were to perform a economic analysis and the influence of atorvastatin, simvastatin, bezafibrate or ciprofibrate on the cardiovascular morbimortality in individuals who used these drugs during the year of 2007, dispensed by the Outpatient Pharmacy of Clinical Hospital of FMRP-USP according to the Exceptional Medicines Program of Ministry of Health. This is an observational and descriptive study of transversal character. The sample was composed of 332 (31,11%) individuals, randomly selected among 1067 patients (standard error of 5%), of both sexes, living in Ribeirão Preto-SP conveyed by the Single System of Health (SUS) and private clinics. Individuals were submitted to an interview and had their medical records examined. Among the 310 patients interviewed, 157 (51%) were males with ages ranging from 15 to 63 years old (X= 62,0 ± 12,23). Five deaths were reported in 2007, and of those patients, 100% were males, with ages ranging from 57 to 74 years old (X= 68,2 ± 6,95). 227 (73,22%) patients were using statins, 54 (17,42%) fibrates and 31 (10%) controls (no use of drugs). The average of body mass index (BMI) of 246 (79,35%) patients evaluated was above 28,7 Kg/m2; 121 (39%) patients were using simvastatin, 104 (34%) atorvastatin, 25 (8%) ciprofibrate and 29 (9%) bezafibrate. The lipid profile was more elevated in atorvastatin and bezafibrate groups. A total of 253 events and/or procedures were found. 132 (52,17%) patients had atherosclerosis documented, 60 (23,71%) angina pectoris, 28 (11,47%) heart failure, 6 (2,44%) acute myocardial infarction, 6 (2,44%) arterial aneurysm, 4 (1,62%) vascular brain accident. Regarding the procedures, 11 cardiac catheterism and 7 angioplasties were verified. Regarding the economic analysis, atorvastatin treatment group showed to be the most expensive one (R$ 994,69 patient/year). For the simvastatin group (R$337,61 patient/year), there were increased costs for lab and complementary tests, while among the group of fibrates there were no substantial differences in the cost of treatments. It is concluded that among the evaluated individuals, there was a prevalence of elderly people, deaths of male patients and overweight (BMI > 25kg/m2). The presence of atherosclerosis and angina pectoris were the predominant cardiovascular events and the cardiac catheterism procedure was the most performed. Although treatment with atorvastatin was the most expensive, patients in that treatment had a lower incidence of cardiovascular events and procedures, and lower costs with lab and complementary tests.

Orientador: Maria Helena de Melo Lima / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Enfermagem / Made available in DSpace on 2018-08-23T13:59:27Z (GMT). No. of bitstreams: 1 Banhesse_VanessaFerrazSuzuki_M.pdf: 2526761 bytes, checksum: 40666dc2d8f53b2d7f50666126a76702 (MD5) Previous issue date: 2013 / Resumo: A cicatrização de feridas em pele é um processo biológico complexo e dinâmico, que envolve eventos como inflamação, proliferação, e remodelamento. Estudos recentes mostraram que estatinas, por meio de efeitos pleiotrópicos independentes da ação hipolipemiante, são novas opções terapêuticas para tratamento de diferentes patologias por suas ações anti-inflamatórias, antioxidantes, sobre vasodilatação, neoangiogênese e na redução da disfunção endotelial. O objetivo deste estudo foi investigar a ação da atorvastatina no reparo tecidual em lesões agudas em animais saudáveis. Os animais foram distribuídos em quatro grupos: placebo (P); atorvastatina tópica (AT); atorvastatina oral (AO); atorvastatina tópica e oral (ATO). Sob anestesia, a lesão na região dorsal foi confeccionada com punch de 8mm. As lesões foram fotografadas nos dias 0, 1, 3, 7, 10 e 14 pós-lesão e amostras destas extraídas nos dias 1, 3, 7 e 14, para análise da expressão de proteínas PI3K, Akt, GSK-3, eNOS, VEGF, TGF-?, ERK, IL- 10, IL-1?, IL-6 e TNF-?. Na avaliação macroscópica, houve melhora significativa quanto à redução das áreas das lesões nos grupos AT, AO e ATO em relação ao placebo. Na avaliação da expressão de proteínas, no 1o dia houve aumento da expressão PI3K, Akt, GSK-3, eNOS e IL-10 nos grupos AT e AO, em relação ao placebo. No 3o dia, houve aumento da expressão das proteínas PI3K, Akt, GSK-3 e IL-10 e no 7o dia, houve aumento da expressão da PI3K, Akt, GSK-3, IL-10, eNOS, VEGF e ERK em todos os grupos tratados com atorvastatina, em relação ao placebo. No 1o, 3o e 7o, houve redução na expressão das citocinas IL-6 e TNF-? em todos os tratados em relação ao placebo. Concluímos que atorvastatina acelera o reparo tecidual em modelo de lesões agudas em ratos e modula a expressão das proteínas nas vias de crescimento celular e citocinas / Abstract: Wound healing in skin is a complex and dynamic biological process, involving events such as inflammation, proliferation, and remodeling. Recent studies have shown that statins, through pleiotropic effects independent of its lipid lowering action, are new therapeutic options for the treatment of different diseases for their anti-inflammatory and antioxidant actions, on vasodilation and neoangiogenesis and reduction of endothelial dysfunction. The aim of this study was to investigate the effect of atorvastatin in tissue repair after acute injury in healthy animals. The animals were divided into four groups: placebo (P); topical atorvastatin (AT); oral atorvastatin (AO); topical and oral atorvastatin (ATO). Under anesthesia, a lesion in the dorsal region in the animal was made with 8mm punch. The lesions were photographed on days 0, 1, 3, 7, 10 and 14 post-injury and the samples drawn on days 1, 3, 7 and 14 for protein expression analysis of PI3K, Akt, GSK-3, eNOS VEGF, TGF-?, ERK, IL-10, IL-1?, IL-6 and TNF-?. At macroscopic examination, there was significant improvement in the reduction of the lesion areas in groups AT, AO and ATO relative to placebo. Evaluating the expression of proteins on day 1, there was an increased expression of PI3K, Akt, GSK-3, eNOS and IL-10 in AT and AO groups, when compared to placebo. At day 3, there was increased expression of PI3K, Akt, GSK-3 and IL-10 and in the day 7, there was increased expression of PI3K, Akt, GSK-3, IL-10, eNOS, VEGF and ERK in all atorvastatin-treated groups versus placebo. In the days 1, 3 and 7, there was a reduction in the expression of IL-6 and TNF-? in all atorvastatin-treated versus placebo. We conclude that atorvastatin accelerates tissue repair in a model of acute lesions in rats and modulates expressions of proteins in cell growth pathways and cytokines / Mestrado / Enfermagem e Trabalho / Mestra em Ciências da Saúde

Abstract Enzymes catalyze chemical changes in biological systems. Therefore, to understand the chemistry of living systems, it is important to understand the enzyme structure and the chemistry of the enzyme's functional groups which are involved in catalysis. In this study, structure and function relationships of two enzymes, (1) α-methylacyl-CoA racemase from Mycobacterium tuberculosis (MCR) and (2) argininosuccinate lyase from Escherichia coli (eASL) have been studied using X-ray crystallography. The main focus of this study has been understanding the structure-function relationship of MCR. The eASL has been crystallized from a highly concentrated sample of purified recombinant α-methylacyl-CoA racemase in which it occurred as a minor impurity. The structure of eASL has been solved using molecular replacement at 2.44 Å resolution. The enzyme is a tetramer, but in this crystal form there is a dimer in the asymmetric unit. Each active site is constructed from loops of three different subunits. One of these catalytic loops, near residue Ser277 and Ser278, has been disordered in the previous structures of active lyases, but is very well ordered in this structure in one of the subunits due to the presence of two phosphate ions in the respective active site cavity. The positions of these phosphate ions indicate a plausible mode of binding of the succinate moiety of the substrate in the competent catalytic complex and therefore this structure has provided new information on the reaction mechanism of this class of enzymes. α-Methylacyl-CoA racemase (Amacr) catalyzes the racemization of α-methyl-branched CoA esters. An Amacr homologue from the eubacteria Mycobacterium tuberculosis, referred to as MCR, was taken as a model protein. MCR was purified, crystallized and the structure of unliganded protein was determined at 1.8 Å resolution using the MIRAS procedure. The structure shows that the enzyme is an interlocked dimer. To understand the reaction mechanism and the mode of substrate binding, several crystallographic binding studies were done using both wild type MCR and mutant H126A MCR crystals. In particular, the structures of the wild type MCR-complexes with (R, S)-ibuprofenoyl-CoA (1.85 Å), (R)-2-methylmyristoyl-CoA (1.6 Å) and (S)-2-methylmyristoyl-CoA (1.7 Å) were important in this respect. These crystal structures show that Asp156 and His126 are the two catalytic residues which are involved in proton donation and abstraction, respectively; when the (S)-enantiomeric substrate is bound in the active site and vice versa when the (R)-enantiomeric substrate is bound. The tight geometry of the active site also shows that His126 and Asp156 are involved in stabilizing the transition state. These crystal structures show that in the active site of MCR, there is one binding pocket for the CoA part and there are two different binding pockets (R-pocket and S-pocket) connected by a hydrophobic methionine rich surface for binding the fatty acyl part of the substrate. After substrate binding, proton abstraction takes place which produces a planar intermediate. Then, donation of a proton to the other side of the planar intermediate changes the configuration at the chiral center. During the stereochemical interconversion of the two enantiomers, the acyl group moves between R-pocket and S-pocket by sliding over the hydrophobic surface connecting these two pockets.

Les rhabdomyolyses correspondent à la destruction de fibres musculaires striées squelettiques et mettent en jeu le pronostic vital. La principale cause génétique est liée à un défaut d’oxydation des acides gras ; néanmoins, plus de la moitié des cas n’ont pas de cause identifiée. En 2008, des mutations du gène LPIN1 ont été rapportées comme une nouvelle étiologie de rhabdomyolyse de transmission autosomique récessive. La protéine lipin1 a une double fonction : un rôle de PAP1 intervenant dans la synthèse du triacylglycérol et des phospholipides membranaires ; un rôle de co-activateur transcriptionnel en association avec les PPARs et PGC1α pour réguler de nombreux gènes impliqués dans le métabolisme, dont certains de l’OAG. Lipin1 a deux homologues, lipin2 et lipin3, qui possèdent une activité PAP1 et un site de fixation à des récepteurs nucléaires tels que les PPARs. Nous avons montré que les mutations de LPIN1 rendent compte de plus de 50% des cas de rhabdomyolyse sévère de la petite enfance, une fois écarté le diagnostic de déficit de l’OAG. Une délétion intragénique en phase a été fréquemment identifiée chez les Caucasiens. Nous avons montré qu’il s’agissait d’un probable effet fondateur et que cette délétion est délétère. En effet, à l’inverse de la forme normale de lipin1, la forme délétée est incapable de complémenter la levure pah1, déficiente pour l’homologue de LPIN1. Nous avons ensuite étudié, dans une série de 171 patients, l’implication de LPIN1 dans des pathologies musculaires moins sévères, ainsi que le rôle des deux homologues LPIN2 et LPIN3. Les mutations de LPIN1 sont impliquées dans les rhabdomyolyses sévères et précoces uniquement et les accès de rhabdomyolyse ont toujours un facteur déclenchant, le principal étant les infections aiguës fébriles. Aucune altération majeure de LPIN2 et de LPIN3 n’a été identifiée, même dans des phénotypes modérés. Enfin, nous avons cultivé des myoblastes et des myotubes de patients avec mutations de LPIN1 afin d’étudier les mécanismes de rhabdomyolyse. Les myoblastes déficients en lipin1 ont une activité PAP1 très diminuée et une accumulation de gouttelettes lipidiques. Le niveau d’expression des gènes cibles des facteurs de transcription co-activés par lipin1 (PPARδ, PPARα, PGC1α, ACADVL, CPT1B and CPT2) sont inchangés par rapport aux contrôles, alors que le niveau de lipin2 est augmenté. L’analyse transcriptomique sur cultures de myotubes a identifié chez les patients 19 gènes sous-exprimés et 51 sur-exprimés, notamment ACACB, qui code pour Accβ, enzyme clé de la balance synthèse d’acides gras/OAG. L’invalidation d’ACACB par siRNA dans des myoblastes déficients en lipin1 diminue le nombre de gouttelettes lipidiques, confirmant le lien entre la sur-expression d’ACACB et l’accumulation d’acides gras libres chez les patients. Cependant, le taux de malonyl-CoA, produit d’Accβ, et l’activité CPT1 (étape limitatrice de l’OAG, inhibée par le malonyl-CoA), sont comparables entre myoblastes de patients et de contrôles. Néanmoins, le traitement des cultures par l’association de tumor necrosis factor alpha et d’interleukine-1 β, choisis pour simuler les conditions pro-inflammatoires des infections aiguës, entraîne une augmentation encore plus poussée du taux de malonyl-CoA, une diminution de l’activité CPT1 et une augmentation de l’accumulation de gouttelettes lipidiques chez les patients. Au total, nos données placent LPIN1 comme une cause importante de rhabdomyolyse héréditaire. Le déficit en lipin1 entraine une perturbation du métabolisme lipidique, via une sur-expression d’ACACB, qui est exacerbée en conditions pro-inflammatoires. Nos résultats suggèrent que les conséquences du déficit en lipin1 sont compensées par des mécanismes d'adaptation suffisants en condition normale, mais insuffisants pour la demande métabolique induite par des stress environnementaux comme l'infection, conduisant aux rhabdomyolyses. / Rhabdomyolyses correspond to the destruction of skeletal muscular fibers and are possibly life-threatening. The main genetic cause is linked to defects of fatty acid oxidation (FAO) ; nevertheless, half of the cases have no identified aetiology. In 2008, mutations of LPIN1 gene have been reported as a new cause of autosomal recessive rhabdomyolysis. Lipin1 protein has a double function : 1) a role of phosphatidate phosphatase 1 (PAP1) involved in synthesis of triacylglycerol and membrane phospholipids ; 2) a role of transcriptional co-activator which regulates, in association with the PPARs (peroxysome-proliferator activated receptor) and PGC1α (PPARγ-coactivator1α), numerous genes involved in the metabolism including some genes encoding FAO enzymes. Lipin1 has got two homologues, lipin2 and lipin3, which have a PAP1 activity and a binding site for nuclear receptors, such as the PPARs. We have shown that LPIN1 mutations account for more than 50% of the cases of severe rhabdomyolysis of early infancy, when FAO defects have been excluded. An intragenic in frame deletion has been frequently identified in Caucasians. We have shown that it probably comes from a founding effect and that this deletion is deleterious. Unlike normal lipin1, deleted lipin1 protein is unable to complement the Δpah1 yeast which is defective for the yeast LPIN1 homolog. In a series of 171 patients, we have further studied the involvement LPIN1 in less severe muscular diseases, as well as the role of the two homologues LPIN2 and LPIN3. LPIN1 mutations are involved only in severe and early rhabdomyolyses and the bouts of rhabdomyolysis always have a triggerring factor, mainly acute febrile infections. No major alteration of LPIN2 and LPIN3 has been identified, even in milder phenotypes. Eventually, we have cultivated myoblasts and myotubes of patients with LPIN1 mutations in order to study the mechanisms of the rhabdomyolysis. Lipin1-deficient myoblasts have a drastically decreased PAP1 activity and an accumulation of lipid droplets. The expression level of target genes of the transcription factors co-activated by lipin1 (PPARδ, PPARα, PGC1α, acyl-Coenzyme A very long chain dehydrogenase (ACADVL), carnitine palmitoyl-transferase 1B and 2 (CPT1B and CPT2)) are similar to controls, whereas the level of lipin2 is increased. Transcriptomic analysis of myotube cultures have identified in patients 19 under-expressed genes and 51 over-expressed ones, notably ACACB, which encodes Accβ (acetyl-CoA carboxylase β), key enzyme of the balance between fatty acid synthesis and FAO. ACACB invalidation by siRNA in lipin1-deficient myoblasts decreases the number of lipid droplets, comforting the link between ACACB over-expression and free fatty acid accumulation in patients. However, the level of malonyl-CoA, product of Accβ, and CPT1 activity (limitative step of FAO, inhibited by malonyl-CoA), are similar between myoblasts of patients and controls. But treatment of the cultures with an association of tumor necrosis factor α and interleukin-1 β (TNFα + IL-1β), chosen for mimicking pro-inflammatory conditions of acute infections, leads to a further increase of the level of malonyl-CoA, a decrease of CPT1 activity and an increase of lipid droplets accumulation in patients. In total, our data show that LPIN1 is an important cause of inherited rhabdomyolysis. Lipin1 deficiency leads to a disturbance of the lipidic metabolism, via ACACB over-expression, which is exacerbated in pro-inflammatory conditions. Our results suggest that the consequences of lipin1 deficiency are counterbalanced by adaptative mechanisms which are sufficient at basal state, but insufficient for the metabolic request induced by environmental stresses, such as infections, leading to the rhabdomyolyses. Next step is the study of adipose tissue and the establishment of the inflammatory signature of the patients, in order to determine if this new disease is an auto-inflammatory pathology.

Abstract The hydratase/isomerase superfamily consists of enzymes having a common evolutionary origin but acting in a wide variety of metabolic pathways. Many of the superfamily members take part in β-oxidation, one of the processes of fatty acid degradation. One of these β-oxidation enzymes is the Δ3-Δ 2-enoyl-CoA isomerase, which is required for the metabolism of unsaturated fatty acids. It catalyzes the shift of a double bond from the position C3 of the substrate to the C2 position. In this study, the Δ 3-Δ 2-enoyl-CoA isomerase from the yeast Saccharomyces cerevisiae was identified, overexpressed as a recombinant protein and characterized. Subsequently, its structure and function were studied by X-ray crystallography. The yeast Δ 3-Δ 2-enoyl-CoA isomerase polypeptide contains 280 amino acid residues, which corresponds to a subunit size of 32 kDa. Six enoyl-CoA isomerase subunits assemble to form a homohexamer. According to structural studies, the hexameric assembly can be described as a dimer of trimers. The yeast Δ 3-Δ 2-enoyl-CoA isomerase is located in peroxisomes, the site of fungal β-oxidation, and is a necessary prerequisite for the β-oxidation of unsaturated fatty acids; the enoyl-CoA isomerase knock-out was unable to grow on such carbon sources. In the crystal structure of the yeast Δ 3-Δ 2-enoyl-CoA isomerase, two domains can be recognized, the N-terminal spiral core domain for catalysis and the C-terminal α-helical trimerization domain. This overall fold resembles the other known structures in the hydratase/isomerase superfamily. Site-directed mutagenesis suggested that Glu158 could be involved in the enzymatic reaction. Structural studies confirmed this, as Glu158 is optimally positioned at the active site for interaction with the substrate molecule. The oxyanion hole stabilizing the transition state of the enzymatic reaction is formed by the main chain NH groups of Ala70 and Leu126. The yeast Δ 3-Δ 2-enoyl-CoA isomerase hexamer forms by dimerization of two trimers, as in the other superfamily members. An extensive comparison of the five known structures of this family showed that the mode of assembly into hexamers is not a conserved feature of this superfamily, since the distance between the trimers and the orientation of the trimers with respect to each other varied. Marked differences were also detected between the two yeast enoyl-CoA isomerase crystal forms used in this study, one being crystallized at low pH and the other at neutral pH. The results suggest that the yeast Δ 3-Δ 2-enoyl-CoA isomerase could occur as a trimer at low pH.

No description available.

Acetyl-CoA carboxylase (ACC) catalyzes the formation of malnoyl-CoA, which in turn controls the rate of fatty acid metabolism. ACC beta or 2 has been shown to be localized on the mitochondria in close proximity to carnintine palmitoyl transferase 1 (CPT-1), the enzyme responsible for the influx of acyl-CoA into the matrix where beta oxidation takes place. CPT-1 is inhibited by malonyl-CoA produced by ACC. It has been well documented that AMP activated kinase (AMPK) when activated phosphorylates and inactivates ACC. ACC is controlled allosterically by citrate, which activates, and by palmitoyl-COA, which inhibits. In this study, we asked the question, "Does phosphorylation by AMPK effect the inhibition of ACC by palmitoyl-CoA?" ACC was isolated and then subjected to phosphorylation and activity was measured in varying concentrations of acetyl-CoA and citrate. Phosphoryation reduced the substrate (acetyl-CoA) saturation activity curves for ACC at all levels of palmitoyl-CoA. The Ki for palmitoyl-CoA inhibition of ACC was reduced from 1.7 ± 0.25 µM to 0.85 ± 0.13 uM (p<0.05) as a consequence of phosphorylation. In addition the citrate activation curves for ACC were greatly reduced in the presence of palmitoyl-CoA. The data show that skeletal muscle ACC or ACC-beta is more potently inhibited by palmitoyl-CoA after phosphorylation by AMPK. During long-term exercise when AMPK is activated and muscle palmitoyl-CoA is elevated this may contribute to the low malonyl-CoA and increased fatty acid oxidation.