Utilize este identificador para referenciar este registo: http://hdl.handle.net/10451/20476
Título: S-adenosylhomocysteine promotes endothelial dysfunction and activation : a role for hypomethylation in vascular disease
Autor: Barroso, Maria Madalena H. Serras Vicente, 1986-
Orientador: Castro, Rita Azevedo e, 1963-
Handy, Diane Elizabeth
Palavras-chave: Teses de doutoramento - 2015
Data de Defesa: 2015
Resumo: omocysteine has been established as a risk factor for cardiovascular disease (CVD) by mechanisms incompletely defined. S-Adenosylhomocysteine (SAH) is the metabolic precursor of homocysteine that accumulates in the setting of hyperhomocysteinemia and is a negative regulator of most cell methyltransferases. This thesis project investigated whether methylation imbalance, caused by excess SAH, disrupts endothelium homeostasis and favors the establishment of a pro-atherogenic phenotype. To experimentally address this possibility, studies were conducted in human endothelial cells, in which SAH accumulation was induced using either a pharmacologic or a siRNA approach. As the major regulator of vascular homeostasis, the endothelium exerts a number of vasoprotective effects that are largely mediated by nitric oxide (NO), the most potent endogenous vasodilator. Decreased NO bioavailability is a principal manifestation of underlying endothelial dysfunction, an early marker of atherosclerosis and CVD. To determine whether excess SAH alters NO bioavailability, the expression and activity of endothelial nitric oxide synthase (eNOS), and NO production were monitored in cells. These experiments showed that excess SAH increased the levels of eNOS mRNA but caused a decrease in eNOS protein and activity, to decrease cellular production of NO. Another important feature of endothelial dysfunction is oxidative stress. Studies in endothelial cells revealed that a hypomethylating environment, induced by excess SAH, impairs, not only NO production, but also the cellular redox state. Glutathione peroxidase-1 (GPx-1) is a selenoprotein and a major cellular antioxidant. A link between homocysteine-associated suppression of GPx-1 and endothelial dysfunction had been reported previously; however, the causal molecular mechanisms remained unresolved. The experiments presented here demonstrate a specific mechanism by which SAH-mediated hypomethylation suppresses GPx-1 expression and leads to inflammatory activation of endothelial cells. The expression of a subset of selenoproteins (including GPx-1) is dependent on a specific methylation of the selenocysteine-tRNA (Sec-tRNA). Thus, SAH accumulation was found to inhibit the formation of this methylated isoform of Sec-tRNA resulting in decreased GPx-1 expression, as well as alterations in the expression of other selenoproteins, to promote oxidative stress and a pro-inflammatory activation state in endothelial cells. The observation that Sec-tRNA methylation is decreased by excess SAH, suggests that other RNA species may also be targets for SAH-mediated hypomethylation. Therefore, the effect of SAH on methylation modifications was determined in total and size-fractionated RNA samples from our cell model. Additionally, to confirm these observations, RNA methylation was analyzed in tissue samples from a hyperhomocysteinemic mouse model, where SAH accumulation results from a genetic disorder affecting homocysteine metabolism. Conditions of excess SAH altered the content of some RNA methylation modifications, suggesting that specific RNA methyltransferases may be more susceptible to inhibition by SAH. The activation of endothelial cells that occurs during atherogenesis is characterized by the up-regulation of adhesion molecules, which by recruiting circulating leukocytes favor their transendothelial migration. In a series of studies, the physiological relevance of SAH-induced endothelial cell activation was demonstrated by determining that these SAH-activated cells promoted leukocyte adhesion and migration. Further, the role of DNA hypomethylation on the SAH-induced up-regulation of adhesion molecules was examined. ICAM-1 (intercellular adhesion molecule 1) was found to be up-regulated by SAH accumulation as well as by a DNA methyltransferase inhibitor, suggesting that its expression may be regulated by DNA methylation. Analysis of its promoter methylation; however, showed that it was demethylated in untreated cells, suggesting that it may be regulated by factors other than DNA promoter methylation in response to excess SAH. To understand better the factors involved in the pro-inflammatory activation of endothelial cells, the role of NFkB (nuclear factor kappa B) in SAH-induced responses was examined. These studies establish a role for NFkB in the endothelial cell response to SAH and further link these responses to a suppression of the epigenetic regulator EZH2 (enhancer of zeste homolog 2). EZH2 is a methyltransferase that regulates gene expression by mediating a repressive histone methylation. These results identify EZH2 as a new target of SAH regulation important in inflammatory responses, demonstrating that EZH2 suppression and NFkB activation mediated by excess SAH accumulation may contribute to its adverse effects in the vasculature. Overall, these studies implicate SAH as a key modulator of epigenetic mechanisms by compromising RNA, DNA, and histone methylation. More importantly, our results clearly present SAH as a key player in the disruption of endothelial homeostasis, supporting a role for SAH as an important mediator of homocysteine-associated vascular disease.
Descrição: Tese de doutoramento, Farmácia (Biologia Celular e Molecular), Universidade de Lisboa, Faculdade de Farmácia, 2015
URI: http://hdl.handle.net/10451/20476
Designação: Doutoramento em Farmácia
Aparece nas colecções:FF - Teses de Doutoramento

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