Crossroads of homocysteine, nitric oxide and asymmetric dimethylarginine metabolisms:involvement of S-adenosylhomocysteine and impaired cellular methylation

Media type: E-Book

Title: Crossroads of homocysteine, nitric oxide and asymmetric dimethylarginine metabolisms:involvement of S-adenosylhomocysteine and impaired cellular methylation

Contributor: Rocha, Mónica Eunice dos Santos [Author]

Published: [Erscheinungsort nicht ermittelbar]: [Verlag nicht ermittelbar], 2012

Language: English

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University thesis: Dissertation, 2012

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Description: Tese de doutoramento, Farmácia (Bioquímica), Universidade de Lisboa, Faculdade de Farmácia, 2012 ; Elevated homocysteine, or hyperhomocysteinemia, is an independent risk factor for vascular disease. However, the precise mechanisms underlying this association, although intensively studied, are still incompletely solved. The precursor of all homocysteine produced in the body is S-adenosylhomocysteine (AdoHcy), a strong methyltransferase inhibitor. When homocysteine accumulates, AdoHcy accumulates as well, potentially disturbing most of the transmethylation processes within the cell. Notably, the role of AdoHcy has gained increased attention, regarding the pathophysiology of hyperhomocysteinemia. In fact, in recent studies, not homocysteine, but rather AdoHcy emerged as a more insightful indicator of vascular disease and tissue damage. Thus, in the present work, we postulate that elevated homocysteine itself may not be the major causative factor for the development of vascular disease, and sought to investigate whether AdoHcy accumulation, by disturbing cellular transmethylation reactions, would influence vascular homeostasis. Specifically, we will focus on the endothelium production of nitric oxide (NO), a potent anti-atherogenic molecule, and on the metabolism of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of the endothelial nitric oxide synthase (eNOS). Chapter 1 comprises a general introduction on homocysteine metabolism, including a review of the major concepts concerning regulation, determinants of hyperhomocysteinemia and proposed pathophysiological vascular mechanisms, with special interest on the NO/ADMA pathways. Chapter 2 presents the aims and outline of this thesis. We first investigated the impact of an impaired methylation environment due to AdoHcy accumulation on NO bioavailability. For that, we cultured human umbilical vein endothelial cells (HUVEC) with adenosine-2,3-dialdehyde (ADA), a well-known inhibitor of the enzyme that reversibly converts AdoHcy in homocysteine, to elevate the intracellular levels of AdoHcy (but not of homocysteine), thus establishing our work model characterized by cellular hypomethylation. We observed a decreased NO production due to lower protein levels and activity of the enzyme eNOS, but yet up-regulated levels of the correspondent mRNA. Taken together, these data suggest that eNOS expression is target of post-translational mechanisms possibly involving methylation steps, hence influenced by AdoHcy (Chapter 3). Since the analysis of global DNA methylation was a central point in this work, we compared two most frequently used methods for measuring global DNA methylation, available in our laboratory: a traditional assay using methylation-sensitive enzyme digestion, and other involving DNA acid hydrolysis. Data confirmed that both methods are feasible tools for DNA methylation studies, but the latter approach presented considerable advantages, not only by allowing an absolute quantification, but also because displayed lower intraday variability (Chapter 4). The next objective was to evaluate the effect of AdoHcy and impaired cellular hypomethylation on ADMA metabolism, namely upon its metabolic degradation by the enzyme dimethylarginine dimethylaminehydrolase (DDAH) (Chapter 5). HUVEC were cultured with ADA or 5-aza-2-deoxycitidine (AZA), which targets DNA methyltransferases and specifically inhibits DNA methylation without accumulation of AdoHcy. Although both compounds induced global DNA hypomethylation, our data showed that only ADA (not AZA) increased DDAH activity, suggesting that AdoHcy stimulates the activity of DDAH by a mechanism independent of DNA methylation. In Chapter 6 we aimed to determine the extent to which protein arginine methylation studies, but the latter approach presented considerable advantages, not only by allowing an absolute quantification, but also because displayed lower intraday variability (Chapter 4). The next objective was to evaluate the effect of AdoHcy and impaired cellular hypomethylation on ADMA metabolism, namely upon its metabolic degradation by the enzyme dimethylarginine dimethylaminehydrolase (DDAH) (Chapter 5). HUVEC were cultured with ADA or 5-aza-2-deoxycitidine (AZA), which targets DNA methyltransferases and specifically inhibits DNA methylation without accumulation of AdoHcy. Although both compounds induced global DNA hypomethylation, our data showed that only ADA (not AZA) increased DDAH activity, suggesting that AdoHcy stimulates the activity of DDAH by a mechanism independent of DNA methylation. In Chapter 6 we aimed to determine the extent to which protein arginine methylation status is affected by accumulation of AdoHcy. For that, protein-incorporated ADMA levels were quantified in our cell model. Interestingly, the data indicated that protein methylation is more sensitive to AdoHcy accumulation than DNA methylation, pinpointing a possible new player in homocysteine-related diseases. In fact, protein methylation is emerging as crucial to cellular homeostasis, and its dysregulation may lead to disease. In Chapter 7, using a diet-induced hyperhomocysteinemia rat model, we investigated the in vivo effect of chronic hyperhomocysteinemia on tissue global DNA methylation. We verified that diets capable of inducing hyperhomocysteinemia also disturb cellular methylation potential in liver and heart, but only severely elevated AdoHcy levels affect global DNA methylation, and in a tissue-specific manner. Increased levels of ADMA have been suggested to contribute for the homocysteine-induced vascular toxicity. CBS (cystathionine β-synthase) deficiency, or Classical Homocystinuria, is an inherited disorder of homocysteine metabolism that results in abnormally elevated levels of homocysteine in the blood, and serious complications in the cardiovascular system. In Chapter 8 we present our study showing that severe hyperhomocysteinemia is not associated with increased ADMA plasma levels in CBS deficient patients, supporting the possibility that intracellular accumulation of AdoHcy, as result of high homocysteine levels, impairs protein methylation processes, thereby the synthesis of ADMA. Lastly, Chapter 9 presents a general discussion, major conclusions and future perspectives disclosed by this thesis. In summary, the work here described provides evidence to what extent a disturbed cellular methylation due to elevated AdoHcy may contribute to the pathophysiology of homocysteine. ; A doença vascular é a principal causa de morbilidade e de mortalidade no mundo ocidental industrializado. Ao longo das últimas décadas, níveis elevados de homocisteína, ou hiper-homocisteinémia (HHcy), têm sido considerados um factor de risco independente para esta patologia. A diminuição da biodisponibilidade do óxido nítrico (NO), metabolito crucial para a homeostasia vascular, é uma manifestação-chave da disfunção endotelial que precede a aterosclerose, e um fenótipo consistentemente associado aos estados de HHcy. Contudo, os mecanismos pelos quais níveis aumentados de homocisteína induzem a disfunção endotelial, apesar de intensamente estudados, não se encontram completamente elucidados. A S-adenosil-homocisteína (AdoHcy) é o precursor metabólico de toda a homocisteína produzida no organismo, e um inibidor da maioria das metiltransferases celulares. Por esta razão, tem sido sugerido um mecanismo indirecto para a toxicidade da homocisteína, secundário a uma acumulação de AdoHcy, e a uma possível alteração dos processos celulares de transmetilação. Com efeito, e independentemente da causa subjacente, a níveis elevados de homocisteína corresponderá uma acumulação de AdoHcy, que poderá perturbar os processos de metilação celular, nomeadamente do DNA e das proteínas, daí resultando consequências fisiológicas importantes. Deste modo, o papel da AdoHcy tem vindo a assumir uma relevância crescente na patofisiologia da HHcy. De facto, dados recentes, quer experimentais quer clínicos, sugerem que a AdoHcy possa vir a ser considerada como o biomarcador adicional à Hcy, ou mesmo de excelência, para a susceptibilidade à doença vascular. Neste trabalho, postulamos então que o aumento dos níveis de homocisteína não são directamente o factor causador de doença vascular, mas sim a acumulação de AdoHcy secundária a esse aumento de homocisteína, a qual, por inibição de mecanismos de metilação celular, nomeadamente, da metilação do DNA e/ou das proteínas, poderá influenciar o metabolismo do NO e/ou do seu principal inibidor endógeno, a dimetilarginina assimétrica (ADMA), diminuindo assim a biodisponibilidade de NO e promovendo a disfunção endotelial. Para testar a nossa hipótese de trabalho, usámos como modelo de estudo, numa primeira abordagem, culturas de células endoteliais humanas. Os dados obtidos através destas experiências ex vivo, foram posteriormente confirmados in vivo, nomeadamente em situações de HHcy quer endógena, em doentes com alterações hereditárias do metab...

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