L'Epoxide Hydrolase soluble (sEH) est une enzyme exprimée dans les vaisseaux. Elle possède un domaine hydrolase à l'extrémité COOH-term (sEH-H) qui métabolise des facteurs vasodilatateurs et anti-inflammatoires comme les acides époxyeicosatriénoïques (EETs). Elle possède également un domaine phosphatase à l'extrémité NH2-term (sEH-P) dont le rôle biologique n'est pas totalement élucidé. Afin de comprendre le rôle de sEH-H et sEH-P dans la calcification vasculaire, des anneaux aortiques de rats et des cellules musculaires lisses vasculaires humaines (CMLVh) ont été exposés à des conditions procalcifiantes pendant 7 et 14 jours respectivement. Le N-acétyl-S-farnesyl-L-cystéine (AFC), un inhibiteur de sEH-P, et le trans-4-(4-(3-adamantan-1-yl-ureido)-)cyclohexyloxy) acide benzoïque (t-AUCB), un inhibiteur de sEH-H, ont été utilisés entre 0,1 et 10 μM. En condition procalcifiante, l'AFC réduit de façon dose-dépendante la calcification vasculaire. Au contraire, le t-AUCB augmente de façon dose-dépendante la minéralisation au sein des anneaux aortiques. Une augmentation de l'activité TNAP a été observée dans les surnageants de culture des anneaux aortiques avec le t-AUCB. Sur les anneaux désendothélialisés ou sur les cultures de CMLVh, les inhibiteurs n'ont pas d'effet sur la calcification, soulignant le rôle crucial joué par les facteurs endothéliaux métabolisés par la sEH. L'ensemble de nos résultats montrent que l'inhibition pharmacologique de la sEH-H augmente la calcification vasculaire in vitro en augmentant la biodisponibilité des EETS. Au contraire, l'inhibition de la sEH-P protège contre la calcification vasculaire à travers un mécanisme dépendant de l'endothélium / Expressed in the vasculature, soluble epoxide hydrolase (sEH) exhibits a COOH-terminal hydrolase domain metabolizing endothelial vasodilator and anti-inflammatory factors like epoxyeicosatrienoic acids (EETs) and, a NH2-terminal phosphatase domain whose biological role remains unclear. To assess the role of sEH phosphatase and hydrolase domains in vascular calcification, rat aortic rings and hVSMCs were exposed to procalcifying culture media for 7 and 14 days, respectively. N-acetyl-S-farnesyl-L-cysteine (AFC), an inhibitor of the phosphatase domain, and trans-4-(4-(3-adamantan-1-yl-ureido)-cyclohexyloxy)-benzoic acid (t-AUCB), a hydrolase domain inhibitor, were used at concentrations ranging from 0.1 to 10 μM. Under procalcifying culture condition, AFC significantly and dose-dependently reduces aortic calcification. Conversely, addition of t-AUCB results in a significant and dose-dependent increase in aortic calcification in rats, without modification of tissue viability. A concomitant increase in TNAP activity was observed in supernatants of aortic rings cultured in the presence of t-AUCB. On de-endothelialized aortic rings or hVSMCs cultures, both inhibitors had no significant effect on the calcification process, pointing out the crucial role played by endothelial factors metabolized by sEH in the control of this biomineralization process. Together, our data demonstrates that pharmacological inhibition of sEH hydrolase increases vascular calcification in vitro by majoring the bioavailability of endothelium- derived EETs. Contrarily, the inhibition of sEH phosphatase is protective against vascular calcification through an endothelium-dependent mechanism.

O ácido araquidônico (AA) é precursor na formação dos eicosanoides, que são mediadores lipídicos com uma série de funções na fisiologia e patologia humana. A maioria dos eicosanoides atuam como mediadores pró-inflamatórios e contribuem para o desenvolvimento e proliferação de tumores. Nesta tese foram avaliados dois mediadores: a 15-deoxi-Δ12,14-PGJ2 (15d- PGJ2) e os ácidos Epoxieicosatrienoicos (EETs), ambos apresentam uma atividade oposta a da maioria dos eicosanoides, ou seja, com uma ação anti-inflamatória e antitumoral. Esses dois mediadores distintos da via do AA foram utilizados nesta tese em dois projetos distintos. Primeiro: A 15d- PGJ2 possui uma atividade antiproliferativa e induziu apoptose para diversos tipos de células tumorais, entretanto, o efeito da15d- PGJ2 em células de cancer da tireoide ainda estava desconhecido. Neste sentido, foram cultivadas in vitro células tumorais da tireoide, da linhagem TPC1, e tratadas com diferentes concentrações de 15d- PGJ2 (0 ate 20 μM), as células tratadas demonstraram uma diminuição na proliferação, e aumento na apoptose, e uma diminuição na liberação e expressão relativa de IL-6. Estes resultados em conjunto sugerem que a 15d- PGJ2 pode ser utilizada como uma nova terapia para o cancer da tireoide. Segundo: Os EETs são metabolizados em seus diois pela epóxi hidrolase solúvel (sEH), para manter a estabilidade dos EETs e a sua atividade antiiflamatoria, foi utilizado um inibidor (TPPU) para sEH em um modelo de periodontite induzida por Aggregatibacter actinomycetemcomitans. O tratamento oral com TPPU, assim como o uso de animais sEH Knockout, levou a uma redução na perda óssea acompanhada da diminuição de moléculas osteoclastogenicas como RANK, RANKL e OPG, demonstrando que a inibição farmacológica da sEH pode ter um valor terapêutico na periodontite e doenças inflamatórias que envolvem a reabsorção óssea. / Arachidonic acid (AA) a precursor in the formation of eicosanoids which are lipid mediators with a number of functions in human physiology and pathology. The most of the eicosanoids act as proinflammatory mediators and contribute to the development and proliferation of tumors. In this thesis we evaluated two mediators: 15-deoxy-Δ12,14-PGJ2 (15d- PGJ2) and epoxieicosatrienoic acids (EETs) both act with an opposite activity of most eicosanoids, with an anti-inflammatory and and anti-tumoral action these two distinct mediators from AA pathway were used in this thesis in two different projects. First: 15d- PGJ2, was described that to have an antiproliferative activity and to induce apoptosis in several types of tumor cells however, the effect of 15d- PGJ2 in thyroid cancer cells was unknown in this sense, we tested in vitro cultured thyroid tumor cells, here in TPC1 cells, and treated with different concentrations of 15d- PGJ2 (0 to 20 uM) the treated cells showed a decrease in proliferation and an increase in apoptosis and a decrease in IL-6 release and relative expression. These key results together demonstrate that 15d- PGJ2 can be used as a new therapy for thyroid cancer. Second: The EETs are converted to their diols by soluble epoxy hydrolase (sEH) to maintain the stability of EETs and their anti-inflammatory activity, an inhibitor (TPPU) against was used to sEH in a periodontitis model induced with Aggregatibacter actinomycetemcomitans. The oral treatment in mice with TPPU and sEH Knockout animals showed bone loss reduction accompanied by a decrease in the osteoclastogenic molecules, like RANK, RANKL and OPG, demonstrating that pharmacological inhibition of sEH may have therapeutic value in periodontitis and inflammatory diseases that involve bone resorption. / Tese (Doutorado)