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UNIVERSIDADE DO ALGARVE Expression Profile And UNIVERSIDADE DO ALGARVE Expression profile and pharmacogenetics of genes candidate to play a role in cardiovascular disease Vera Lúcia Martins Dias Doutoramento em Ciências Biológicas Especialidade de Biologia Celular e Molecular 2010 UNIVERSIDADE DO ALGARVE Expression profile and pharmacogenetics of genes candidate to play a role in cardiovascular disease Vera Lúcia Martins Dias Tese orientada pela Professora Doutora Vera Linda Ribeiro Marques Doutoramento em Ciências Biológicas Especialidade de Biologia Celular e Molecular 2010 Acknowledgments Acknowledgements Muitas foram as pessoas que, ao longo destes anos, me apoiaram directa ou indirectamente. A todos os que tornaram este projecto possível quero manifestar o meu sincero agradecimento. À Professora Doutora Vera Ribeiro Marques, quero agradecer a confiança e a oportunidade de realizar este trabalho sob sua orientação e pela forma como o fez ao longo destes anos; além do apoio e incentivo deu-me a oportunidade e liberdade de crescer profissional e cientificamente. Por tudo isso, os meus sinceros agradecimentos. À Ana Luísa, quero agradecer por me ter ajudado ao longo destes anos, por me ouvir e aconselhar, nem sempre apenas sobre assuntos científicos. Muito obrigada pela tua amizade. Ao Jorge, quero agradecer por todas as contribuições dadas e pelo bom ambiente de trabalho que sempre proporcionaste. Á Filomena, à Elsa, à Cátia, que estiveram presentes nos meus primeiros anos no laboratório e me ajudaram em tudo o que precisei. Muito agradecida por me terem recebido e pela vossa amizade. E a todos aqueles que, ao longo destes nos, passaram pelo laboratório, por terem tornado este percurso muito mais agradável, tendo proporcionado sempre um bom ambiente de laboratório. Por fim, não posso deixar de expressar os meus agradecimentos a toda a minha família. Em especial, aos meus pais, por estarem sempre presentes, por me apoiarem em todas as minhas decisões e pela compreensão com que sempre o fizeram. Muito agradecida por acreditarem em mim e por tudo o que me proporcionaram. Sem vocês não teria conseguido. Á minha irmã por me chamar muitas vezes à razão e por todo o apoio que me tem dado, não posso deixar de expressar os meus sinceros agradecimentos. Ao Ricardo por tudo o que és e representas para mim. Muito agradecida por estares sempre do meu lado, mesmo nos momentos mais difíceis, pela tua confiança e apoio incondicional. 3 Contents Contents Aknowledgments 3 Contents 4 Abstract & Keywords 6 Resumo & Palavras-chave 7 Abbreviations 13 Chapter I - Introduction 16 1. General Introduction 17 2. Cholesterol function and metabolism in mammals 18 2.1. Cholesterol synthesis 19 2.2. Bile acid synthesis 20 3. proteins involved in the metabolism and transport of endogenous and exogenous 22 compounds 3.1. Solute carriers (SLC) 22 3.1.1. SLC10 family 23 3.1.2. SLC22 family 25 3.1.3. SLCO family 26 3.2. Cytochrome P450 (CYP) enzymes 28 3.2.1. CYP3 family 30 3.2.2. CYP7 family 30 3.2.3. CYP8 family 32 3.2.4. CYP27 family 32 3.2.5. CYP39 family 33 3.2.6. CYP51 family 33 3.3. ATP-binding cassette (ABC) transporters 34 3.3.1. ABCA family 36 3.3.2. ABCB family 37 3.3.3. ABCC family 38 3.3.4. ABCG family 40 4. Molecular regulation of cholesterol homeostasis 41 4.1. By nuclear receptors 41 4.1.1. LXR 42 4.1.2. FXR 43 4.1.3. PPAR 43 4.1.4. LRH-1 44 4.1.5. ROR 44 4.1.6. HNF4 45 4.1.7. SHP 45 4 Contents 4.1.8. PXR 45 4.1.9. CAR 46 4.2. By other transcription factors 46 4.2.1. SREBP 46 5. Cholesterol trafficking across intestine and liver 47 5.1. Cholsterol flux in the enterocyte 48 5.2. Cholesterol flux in the hepatocyte 48 5.3. Enterohepatic circulation 49 5.4. Reverse transport of cholesterol 49 6. Pathological situations associated to cholesterol 50 7. Genetic variability and response to treatment 51 Chapter II - Aims 52 Chapter III - Results 54 1. Pharmacogenetics of genes candidate to play a role in cardiovascular disease risk 55 and therapeutics Paper I – Polymorphisms in SLCO1B1, SLCO1B3, SLC22A1 and SLC10A1 56 transporters among Caucasians, native Africans and South Americans Paper II - Ethnic differences in the prevalence of polymorphisms in drug 70 metabolizing enzymes involved in cholesterol metabolism Paper III - Inter-ethnic variability in ABC transporters ABCB11, ABCG5 AND 82 ABCG8 2. Expression profile of genes involved in drug metabolism and transport in response 95 to cholesterol Paper IV - The expression of the solute carriers NTCP and OCT-1 is regulated by 96 cholesterol in HepG2 cells Paper V - Effect of cholesterol on the expression of Solute carriers, Cytochrome 102 P450 enzymes and ABC transporters in HepG2 and Caco-2 cells Chapter IV - Discussion 122 1. Pharmacogenetics of genes candidate to play a role in cardiovascular disease risk 123 and therapeutics 2. Expression profile of genes involved in drug metabolism and transport in response 136 to cholesterol Chapter V – Conclusions and Future Perspectives 155 1. Pharmacogenetics of genes candidate to play a role in cardiovascular disease risk 156 and therapeutics 2. Expression profile of genes involved in drug metabolism and transport in response 157 to cholesterol 3. Future Perspectives 158 Chapter VI - References 159 Appendix I 196 5 Abstract & Keywords Abstract Cardiovascular diseases are the major causes of death and morbidity in developed countries. Several risk factors have been associated to cardiovascular diseases, but the molecular mechanisms underlying the alteration of homeostasis are still largely unknown. There is increasing recent evidence that the drug metabolizing enzyme system associated to a number of membrane transport proteins is of particular relevance in relation to cardiovascular diseases, either in biosynthetic or degradative pathways. The overall aim of the present study was to characterize selected genes coding for drug metabolizing enzymes and influx and efflux transporters both from the point of view of pharmacogenetic diversity and from their response to increased cholesterol concentrations. Specifically we a) characterized, in a sample of the Portuguese population, selected genetic polymorphisms in cytochromes P450, influx and efflux transporters that are known to – or may be expected to - affect in vitro or in vivo the metabolism or transport of cholesterol and/or therapeutic drugs; b) evaluated the existence of ethnic variability in the frequency of these polymorphic variants, through the determination of their prevalence in other populations; and c) investigated to which extent the expression of these genes, in liver and intestine derived cells, may be influenced by the accumulation of cholesterol. Most of the polymorphisms analysed present a similar distribution between Portuguese and Colombian populations, but significant differences when compared with the Mozambican population, demonstrating that these polymorphisms are ethnicity-dependent. The expression profiling studies revealed a differential expression pattern for the analysed genes in cell culture models from liver and intestine and a differential response to cholesterol, pointing to significant differences in gene regulation in these tissues. Taken together, the results obtained in this study contribute to a better knowledge of drug metabolism and transport in hypercholesterolemia as well as to understanding the pharmacogenetic variability of the genes involved in these pathways. Keywords: Cytochrome P450 (CYP) Influx transporters (SLC) Efflux transporters (ABC) Cholesterol Pharmacogenetics 6 Resumo & Palavras-chave Resumo As doenças cardiovasculares são a maior causa de morte e morbilidade nos países desenvolvidos. Embora vários factores de risco estejam associados às doenças cardiovasculares, como pressão sanguínea elevada, obesidade, níveis de colesterol elevados, diabetes, stress e tabaco, os mecanismos moleculares subjacentes às alterações da homeostasia envolvidas não são ainda conhecidos. A prevenção das doenças cardiovasculares baseia-se na terapêutica de redução de lípidos, estando diversos fármacos disponíveis para este propósito, como as estatinas, inibidores da absorção de colesterol, resinas de ácidos biliares, fibratos e ácido nicotínico, os quais actuam no organismo em diferentes vias, tendo por isso diferentes modos de acção entre si. Um número crescente de publicações tem vindo a demonstrar que o sistema de enzimas metabolizadores de xenobióticos (citocromos P450), associado a proteínas de transporte membranar, como a superfamília de transportadores dependentes de ATP (transportadores ABC) ou a superfamília de transportadores de influxo (SLC), é de particular relevância em relação às doenças cardiovasculares, tanto em termos de vias de biossíntese, como das vias de catabolismo. O fígado, sendo o principal órgão de destoxificação do organismo, extrai os xenobióticos do sangue através de transportadores membranares especializados, os transportadores de influxo, tornando estes compostos disponíveis para biotransformação pelos enzimas metabolizadores. Assim, os citocromos P450 modificam o substrato, geralmente aumentando a sua solubilidade, facilitando desta forma, a sua eliminação. Os produtos metabólicos são então excretados da célula através dos transportadores de efluxo ABC. Os transportadores de influxo (SLC) são uma superfamília de transportadores que facilita a passagem de solutos ao longo das membranas celulares, encontrando-se presentes em diversos órgãos. Alguns transportadores de influxo apresentam uma expressão elevada no fígado, órgão no qual desempenham um papel crucial na disposição e eficácia de muitos
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