Stroke is a common, multifactorial cardiovascular disease. A stroke event is the result of traditional risk factors (i.e. hypertension, diabetes, smoking), environmental exposures and genetic factors in a complex interplay. The genetic contribution is, as estimated by studies on the influence of family history on the risk of stroke, limited on the individual level, and overridden by, for example the excess risk associated with smoking. On the population level, and as a means to better understand the etiology of stroke, genetics can play a major role. Northern Sweden is well suited for studying the genetic aspects of stroke. The population shows signs of founder effects, and is relatively homogeneous. Large-scale cardiovascular health surveys, the MONICA Project and the Västerbotten Intervention Program, allow studies on risk factors in relation to stroke. Two prospective nested case-referent study samples, (113 cases and 226 controls; 275 cases and 549 controls), and a set of 56 families (117 affected) were collected for functional candidate gene association, and linkage, studies. The selected candidate genes included haemostatic factors and genes within the renin angiotensin system (RAS). Functional single nucleotide polymorphisms (SNPs) that influence the levels of PAI-1 (PAI-1 4G/5G), and tPA (tPA -7,351C>T), have been identified. The angiotensin converting enzyme insertion/deletion polymorphism (ACE I/D) has been shown to be associated with ischaemic stroke. The angiotensin II receptor type 1 A1166C polymorphism (AT1R A1166C), less extensively studied, has been suggested to be associated with stroke, and to interact with the ACE I/D. We found that the PAI-1 4G/4G genotype was associated with an increased risk of future ischaemic stroke (OR 1.79, 95%CI 1.01-3.19), and this was replicated in a second study sample. Furthermore, levels of serum triglycerides modulated the effect of the genotype. In the study on tPA, no association between the tPA -7,351C>T polymorphism and the risk of stroke was found in an analysis of the two study samples pooled. The two RAS polymorphisms were prospectively associated with ischaemic stroke independently of each other and other risk factors (OR 1.60, p=0.02 and OR 1.60, p=0.04, respectively). A candidate region linkage study, focusing on a previously reported stroke susceptibility locus on chromosome 5, was performed in a set of families. In addition, association between ischemic stroke and the positional candidate gene phosphodiesterase 4D (PDE4D) was tested. Linkage to 5q12 was replicated in this independent population, but not PDE4D association with stroke. This suggests that alternative genotypes in this stroke susceptibility locus contribute in different populations. In conclusion, the genetic component in the causation of stroke was investigated. The results of the functional candidate gene association studies showed (1) interaction between PAI-1 genotype and a putatively modifiable risk factor, triglycerides, (2) a prospective testing of the tPA SNP with no association detected, and (3) a novel, hypothesis-generating, finding in the case of AT1R polymorphism and the risk of ischaemic stroke. The replication of linkage to chromosome 5q12 in our northern Swedish population was interesting, and it will be further explored.

L’hypertension systolique isolée (HSI) est le résultat de changements au niveau de la paroi vasculaire qui ont pour conséquence d’augmenter la rigidité artérielle. Ces modifications surviennent surtout au niveau des grosses artères comme l’aorte et sont associées au vieillissement. La fragmentation des fibres élastiques, leur calcification (élastocalcinose) et la fibrose font partie des changements majeurs observés avec l’âge. En plus de ces changements, le vieillissement vasculaire provoque des modifications au niveau des cellules qui composent la paroi. Les cellules endothéliales sécrètent moins de monoxyde d’azote (NO) provoquant une dysfonction endothéliale et les cellules musculaires lisses vasculaires (CMLVs) synthétisent maintenant des protéines matricielles et osseuses. Situé entre le sang et les CMLVs, l’endothélium contrôle le tonus vasculaire par la sécrétion de plusieurs substances vasoactives qui interagissent entre elles afin de maintenir l’homéostasie du système vasculaire. Parmi celles-ci, on note l’endothéline (ET), un puissant vasoconstricteur et le NO, un gaz vasorelaxants. Ce dernier est aussi reconnu pour bloquer la production d’ET par un mécanisme dépendant du guanosine monophosphate cyclique (GMPc). Comme il y a une interaction entre le NO et l’ET, et que cette dernière est impliquée dans la calcification artérielle, le NO pourrait être impliqué dans la modulation de l’élastocalcinose et de la rigidité artérielle par l’inhibition de l’ET et la modification de la composition de la paroi. Cet effet, qui se produirait au delà des effets vasorelaxants du NO, offre un potentiel thérapeutique intéressant pour l’HSI. Afin d’évaluer l’implication du NO dans la calcification vasculaire et la rigidité artérielle, un modèle animal d’HSI a été utilisé (modèle warfarine vitamine K, WVK). Ce modèle d’élastocalcinose est basé sur l’inhibition de la maturation d’une protéine anti-calcifiante, la matrix Gla protein (MGP), par la warfarine. Afin de déterminer l’implication physiologique du NO dans l’initiation et la progression de l’élastocalcinose, sa production a été inhibée par un analogue de la L-arginine, le L-NG-nitroarginine methyl ester (L-NAME). Lors des processus d’initiation de la calcification, le L-NAME a prévenu l’élastocalcinose sans toutefois modifier la vitesse de l’onde de pouls (PWV). Suite au traitement L-NAME, l’expression de la NO synthase inductible (iNOS) a été diminuée alors qu’elle a été augmentée lors du traitement WVK. Elle pourrait donc être impliquée dans les processus de calcification vasculaire. De plus, la NO synthase endothéliale (eNOS) semble également impliquée puisqu’elle a été augmentée dans le modèle WVK. Cette hausse pourrait être bénéfique pour limiter l’élastocalcinose alors que l’expression de la iNOS serait délétère. Lors de la progression de la calcification, le L-NAME a augmenté l’élastocalcinose et le PWV. Dans ce contexte, l’ET serait impliquée dans l’amplification de la calcification vasculaire entrainant une hausse de la rigidité artérielle. Comme le NO endogène limite la progression de la calcification et conséquemment la rigidité artérielle, il semble être protecteur. L’efficacité d’une modulation de la voie du NO dans le modèle WVK a été étudiée par l’administration d’un donneur de NO, le sinitrodil, ou d’un inhibiteur de la phosphosdiestérase 5 (PDE5), le tadalafil. La modulation de la voie du NO semble être bénéfique sur la rigidité artérielle, mais seulement de façon aiguë. En effet, le sinitrodil a modifié de transitoirement la rigidité au niveau de l’aorte possiblement par la modulation du tonus vasculaire sans toutefois avoir des effets sur la composition de la paroi. Comme le modèle WVK n’affecte pas la fonction endothéliale, les concentrations endogènes de NO semblent être optimales puisque le sinitrodil provoque une augmentation de l’élastocalcinose possiblement par le développement d’une tolérance. Tout comme le sinitrodil, le tadalafil a modulé de manière aiguë la rigidité artérielle sans modifier la composition de la paroi. Globalement, ces travaux ont permis de mettre en évidence les effets bénéfiques du NO endogène pour limiter le développement de l’HSI, suggérant qu’une dysfonction endothéliale, tel qu’observé lors du vieillissement, a un impact négatif sur la maladie. / Isolated systolic hypertension (ISH) is the result of complex changes in the vascular wall and consequently the increase of arterial stiffness. These modifications occur mainly in conductance arteries, like the aorta, and are associated with aging. The fragmentation of elastic fibers, calcification (elastocalcinosis), and fibrosis are major changes with age. In addition to these changes in the extracellular matrix, vascular aging also induces vascular cell wall modifications. These include decreased production of nitric oxide (NO) by endothelial cells, which induces endothelial dysfunction, and the production of matrix and bone proteins by vascular smooth muscle cells (VSMCs). Located between the blood and VSMCs, the endothelium controls vascular tone by secreting various vasoactive factors. These factors interact with each other to maintain the hemodynamic of the vascular system. Among these factors, the vasoconstrictor endothelin (ET) and the vasodilator NO. The latter has been shown to block ET production via a cyclic guanosine monophosphates-(cGMP) dependent mechanism, whereas ET has been implicated in arterial calcification. Therefore, NO might be involved in the modulation of elastocalcinosis and arterial stiffness by inhibiting ET and modifying the vascular wall composition. This effect of NO could offer interesting therapeutic potential for ISH. To evaluate the implication of NO in the vascular calcification and arterial stiffness, an animal model of ISH was used. This model of elastocalcinosis is based on the inhibition of the maturation of the anti-calcific protein, matrix Gla protein (MGP), by warfarin (WVK model). To gain insight into the physiological role of endogenous NO in the initiation and progression of elastocalcinosis, its production was inhibited by the administration of L-NAME. Interestingly, elastocalcinosis was prevented by L-NG-nitroarginine methyl ester (L-NAME) administration without any modifications of the pulse wave velocity (PWV) during the initiation of the calcification processes. After the L-NAME treatment, the expression of inducible NO synthase (iNOS) was decreased, whereas upon treatment with warfarin alone the expression of iNOS was increased, which could be implicated in vascular calcification and arterial stiffness. In addition, endothelial NO synthase (eNOS) seems to be implicated in this process as its expression was also increased upon WVK treatment. This increase could be beneficial to limit elastocalcinosis, whereas the increase in iNOS expression could be harmful. L-NAME administration during the progression of calcification increased elastocalcinosis and PWV. In an endothelial dysfunction context, ET has been shown to be involved in the amplification process of vascular calcification causing an increase in arterial stiffness. As NO limits the progression of calcification and consequently arterial stiffness, endogenous NO seems to be protective in the aorta. The efficacy of exogenous modulation of the NO pathway in the WVK model was studied upon administration of the NO donor, sinitrodil, or the phosphodiesterase type 5 inhibitor (PDE5), tadalafil. The exogenous modulation of the NO pathway seemed to be beneficial for arterial stiffness, but only in an acute manner. Indeed, sinitrodil modified the acute stiffness in the aorta potentially by vascular tone modulation, without having any effect on vascular wall composition. Since endothelial function was not affected upon WVK model, endogenous NO concentrations seem to be optimal. Thus, exogenous NO potentially caused an increase of elastocalcinosis by inducing tolerance to NO. As well as sinitrodil, tadalafil modulated the arterial stiffness in an acute manner without modifying the composition of the vascular wall. Broadly, these studies provide evidence that endogenous NO can limit ISH development, suggesting that endothelial dysfunction, as observed in aging, has a negative impact on this pathology.

Introduction: La circulation extracorporelle (CEC) peut entraîner une dysfonction endothéliale pulmonaire et l’hypertension pulmonaire. Le SN50 agit au niveau de la signalisation cellulaire pour prévenir ces réactions à la CEC et pourrait renverser la dysfonction endothéliale pulmonaire post-CEC sans effets néfastes sur l’hémodynamie. Méthodes: Quatre groups de porcs ont reçu un parmi quatre traîtements avant de subir 90 minutes de CEC et 60 minutes de reperfusion: (1) milrinone nébulisé; (2) sildenafil nébulisé; (3) placebo nébulisé; et (4) SN-50 intraveineux. Un monitoring hémodynamique invasif a été utilisé. La réactivité vasculaire des artères pulmonaires de deuxième ordre a été évaluée face à l’acétylcholine et la bradykinine. Résultats: Le sildénafil produit une augmentation significative de la pression de l’artère pulmonaire (PAP) moyenne à 60 minutes de reperfusion par rapport au début de la chirurgie. Les relaxations dépendantes de l’endothélium face à la bradykinine étaient meilleurs dans les groupes milrinone et SN-50 et surtout dans le groupe sildénafil par rapport au groupe placébo. Le SN-50 produisait de moins bonnes relaxations dépendantes de l’endothélium face à l’acétylcholine que les autres traitements incluant placébo. Conclusion: Le sildénafil prévient mieux la dysfonction endothéliale pulmonaire que les autres traitements. Les bénéfices du SN-50 sont possiblement sous-estimés vu que la dose n’a pas pu être ajustée à la durée de CEC. Le sildenafil inhalé mérite une étude plus importante chez l’humain et le SN-50 dans un model de CEC animal. / Background: Cardiopulmonary bypass (CPB) can lead to pulmonary endothelial dysfunction and consequent pulmonary hypertension. The novel agent SN-50 acts at the level of the transduction pathway to prevent these responses and may limit or reverse post-CPB pulmonary endothelial dysfunction and pulmonary hypertension without the untoward effects on hemodynamics seen with other known therapies. Methods: Four groups of Landrace-Yorkshire swine that received one of four treatments before undergoing 90 minutes of normothermic CPB and 60 minutes of reperfusion were compared: (1) Nebulized milrinone; (2) nebulized sildenafil; (3) placebo consisting of nebulized NaCl solution; and (4) intravenous SN-50. Invasive hemodynamic monitoring was used throughout all experiments. Vascular reactivity of second-degree pulmonary arteries was evaluated in response to acetylcholine and bradykinin. Results: Sildenafil produced a significant increase in mean pulmonary artery pressure (PAP) at 60 minutes after CPB compared to baseline. Both the sildenafil and milrinone groups had increased mean PAP/MAP ratio at 60 minutes after CPB compared to baseline, however this ratio was not different between the groups. Endothelial-dependent relaxations to bradykinin were improved in the SN-50 and milrinone groups and especially the sildenafil group as compared to placebo. SN-50 produced worse endothelium-dependent relaxations in response to acetylcholine compared to the other groups including placebo. Conclusion: Sildenafil better prevented pulmonary endothelial dysfunction than all other treatments. The improvements seen with SN-50 may be suboptimal as dose could not be titrated to length of CPB. Inhaled sildenafil and SN-50 both merit further study in human trials and animal models, respectively.

Os pacientes que usam lítio (Li) para tratamento do transtorno bipolar frequentemente apresentam poliúria e deficiência de concentração urinária, sintomas do Diabetes Insipidus Nefrogênico (DIN). Animais tratados com Li apresentam baixos níveis de produção de adenosina monofosfato cíclico (AMPc) em resposta ao hormônio antidiurético (HAD). O Sildenafil (Sil), um inibidor da fosfodiesterase 5 (PDE5), eleva os níveis intracelulares de guanosina monofosfato cíclico (GMPc), levando a inserção de aquaporina 2 (AQP2) na membrana plasmática das células do ducto coletor. Portanto, inibidores de PDE podem promover a inserção de AQP2 na membrana plasmática mesmo sem a ativação do receptor de HAD, indicando a participação de uma via alternativa mediada pelo GMPc. Nós investigamos as vias de ação do Sil no tratamento da DIN induzida pelo Li. Ratos Wistar foram divididos nos seguintes grupos: grupo controle, recebendo dieta alimentar normal durante quatro semanas; grupo Li, recebendo dieta alimentar normal com 40 mmol Li por quilo de dieta durante quatro semanas; grupo Li + Sil, recebendo dieta alimentar normal com 40 mmol Li por quilo de dieta durante quatro semanas e 200 mg por quilo de dieta de Sil a partir da segunda semana; grupo Sil, recebendo dieta alimentar normal durante a primeira semana e a partir da segunda semana recebendo dieta normal com 200 mg de Sil por quilo de dieta. Os animais do grupo Li desenvolveram poliúria, diminuição da osmolalidade urinária e diminuição da expressão da AQP2 tanto na fração citoplasmática como de membrana celular e o Sil reverteu essas alterações. Demonstramos ainda que a concentração de GMPc intracelular estava aumentada nos túbulos papilares tratados com Sil. Observamos que a provável via de fosforilação da AQP2 induzida pelo GMPc é pela PKA. Além disso, o tratamento com Sil aumenta a expressão de pCreb, fator de transcrição para ativação do gene da AQP2. Observamos ainda que o Li diminui a expressão de eNOS e o tratamento com Sil normaliza essa diminuição. Assim, concluímos que o tratamento com Sil em ratos com DIN melhora a poliúria aumentando a produção e a inserção de AQP2. O tratamento com Sil pode ser benéfico para pacientes que sofrem com DIN induzido pelo Li / Patients taking lithium to treat bipolar disorder often present polyuria and urinary concentrating defect. In addition, lithium-treated animals present lower cyclic adenosine monophosphate production in response to vasopressin. Sildenafil (Sil), a phosphodiesterase 5 (PDE5) inhibitor, elevates intracellular cyclic guanosine monophosphate (cGMP) levels, leading to plasma membrane accumulation of aquaporin 2 (AQP2). Therefore, PDE inhibitors might induce AQP2 membrane insertion even without vasopressin receptor activation by activating a parallel cGMP-mediated signal transduction pathway. We investigated Sil pathways of action in rats with lithium-induced nephrogenic Diabetes Insipidus (NDI). Wistar rats received lithium (40 mmol/kg food) or not for 4 weeks (Li or control), some rats also receiving sildenafil (200 mg/kg food) in weeks 2-4, with or without lithium (Li+Sil orSil). Animals in Li group developed polyuria, decreased urinary osmolality and decreased expression of AQP2 in both the cytoplasmic fraction and the cell membrane and Sil reversed these changes. We also demonstrated that intracellular cGMP concentration was increased in papillary tubules treated with Sil. We found that PKA may be involved in the pathway of cGMP induced AQP2 phosphorylation. In addition, Sil treatment increases Creb phosphorylation. Creb phosphorylation, acts as AQP2 gene transcription factor. We also observed that Li decreases eNOS expression and treatment with Sil normalizes this alteration. We conclude that Sil treatment improves polyuria by increasing production and insertion of AQP2. Sil treatment may be beneficial to patients suffering from induced DIN Li

Foram estudadas as enzimas implicadas no metabolismo de cAMP, bem como as variações na concentração deste nucleotídeo cíclico e na atividade de adenilato ciclase durante o ciclo biológico de B. emersonii. Demonstrou-se que os zoósporos contêm enzimas específicas e distintas para a hidrólise de cAMP e cGMP. Existe apenas uma espécie molecular da cAMP fosfodiesterase, que hidrolisa cAMP a 5\'-AMP com um Km aparente de 2-4 µM; a presença de cGMP nas misturas de reação, não altera as propriedades cinéticas da enzima. A adenilato ciclase de B. emersonii é uma enzima particulada, provavelmente ligada à membrana plasmática do zoósporo, que exige especificamente Mn2+ para sua atividade. A enzima não é ativada por NaF, catecolaminas ou outros compostos de estrutura semelhante. O estudo das propriedades cinéticas da adenilato ciclase sugere um modelo simples no qual o verdadeiro substrato da enzima é o complexo MnATP2- e tanto ATP corno Mn2+ , nas suas formas livres, competem com o complexo pelo sítio catalítico da enzima, que apresenta uma afinidade maior pelas formas livres do que pelo complexo MnATP2-. A atividade especifica da adenilato ciclase, determinada durante o ciclo biológico do fungo, mostra-se elevada nos zoósporos, cai lentamente durante a germinação e permanece baixa em todo o período de crescimento, só voltando a apresentar um aumento na atividade após a indução da esporulação. Quando este processo e induzido na presença de cicloheximida, a atividade permanece baixa, sugerindo que a enzima é sintetizada \"de novo\" nesta fase do ciclo evolutivo. A concentração intracelular de cAMP foi também determinada nas várias fases do ciclo biológico de B. emersonii. No zoósporo encontrou-se um valor médio de 33 pmoles cAMP/mg proteína. Durante a germinação, os níveis de cAMP aumentam, atingindo um máximo (~ 100 pmoles/mg proteína)quando a quase totalidade dos zoósporos se transformou em esferócitos. A partir daí observou-se um declínio gradual nos níveis de cAMP, que permanecem baixos durante toda a fase de crescimento, voltando a elevar-se na fase final da esporulação até alcançar o nível de zoósporo. O grande aumento na concentração intracelular de cAMP na fase de esferócitos é parcialmente explicado pela predominância da atividade de adenilato ciclase sobre a atividade de cAMP fosfodiesterase neste período; a possibilidade de uma ativação \"in vivo\" da adenilato ciclase, neste estágio do ciclo, não pode ser excluída. A queda nos níveis de cAMP que ocorre na passagem de esferócito a gérmen, numa fase onde a atividade de cAMP fosfodiesterase já e muito baixa, é devido principalmente a excreção deste nucleotídio cíclico para o meio extracelular. O grande aumento nos níveis de cAMP durante a transição de zoósporo a esferócito pode estar relacionado com a ativação metabólica ocorrendo nesta fase e pode também refletir uma característica de sistemas em diferenciação, isto é, a necessidade de altos níveis de cAMP para a transição entre dois estados celulares diferenciados. / The enzymes involved in the metabolism of cAMP have been studied, as well as the fluctuations in the concentration of this cyclic nucleotide and in the adenylate cyclase activity during the life cycle of B. emersonii. Zoospores were shown to contain independent specific enzymes involved in the hydrolysis of cAMP and cGMP. A single molecular species was found for the cAMP phosphodiesterase activity, which catalyses the hydrolysis of cAMP to 5\'-AMP. This enzyme displays normal Michaelis kinetics with an apparent Km of 2-4 µM; the addition of cGMP to the reaction mixtures does not modify the kinetic properties of the enzyme. Adenylate cyclase activity in B. emersonii is associated with particulate subcellular fractions, most probably bound to the zoospore plasma membrane. The activity requires Mn2+ and it is not activated by NaF, cathecolamines or other related compounds. The enzyme substrate is the MnATP2- complex and the kinetic data obtained studying the adenylate cyclase activity can be explained by a simple model where free ATP and Mn2+ compete with MnATP2- for the catalytic site of the enzyme, the affinity for MnATP2- being lower than for free Mn2+ and ATP. The specific activity of adenylate cyclase has been determined throughout the fungus life cycle. The enzyme activity is high in zoospores, falls slowly during germination remaining low at the growth phase and rising again during the later stage of sporulation. When this process is induced in the presence of cycloheximide, there is no increase in adenylate cyclase activity, suggesting that \"de novo\" synthesis of the enzyme occurs at this stage. Fluctuations in the intracellular levels of cAMP during the cell cycle of B. emersonii have also been shown. Zoospores contain an average concentration of 33 pmoles cAMP/mg protein. During germination, a significant increase in the cAMP levels is observed, reaching a maximum (ca. 100 pmoles/mg protein) when the majority of the zoospores have changed into round cells. From then on a gradual decline in the cAMP levels is observed. During the growth phase the cAMP contents of the cells remain low, increasing again late in the sporulation stage. The large increase in the intracellular concentration of cAMP in the round cell phase is partially explained by the predominance of adenylate cyclase activity over cAMP phosphodiesterase activity (during this stage); the possibility of an \"in vivo\" activation of the adenylate cyclase during this period, however, cannot be excluded. The decrease in the cAMP levels occurring during the passage of round cells to germlings, in a stage where cAMP phosphodiesterase activity is negligible, is mainly due to the excretion of this cyclic nucleotide to the extracellular medium. The rise in cAMP contents during encystment might be related to the activation of metabolism occurring in this phase and may also reflect a characteristic of differentiating systems, that is, high cAMP levels being necessary for a differentiative transition.

This thesis is concerned with synthesis, structure and biochemical analysis of chemically modified oligonucleotides with potential therapeutic applications. The three types of chemical modifications described here are: (a) A North-East locked 1',2'-azetidine nucleoside (b) A North locked 2',4'-cyanomethylene bridged nucleoside and (c) A 2',4'-aza-ENA-T nucleoside. The synthesis of the 1',2'-azetidine fused nucleosides was described using two different approaches. A highly strained 2',4'-cyanomethylene locked nucleoside was synthesized but could not be converted to the phosphoramidite derivative due to instability during derivatization. The key cyclization step in the aza-ENA-T nucleoside synthesis gave rise to two separable diastereomers due to chirality at the exocyclic nitrogen. Conversion of diastereomer 55 to 56 occurred with a large free energy of activation (ΔG‡ = 23.4 kcal mol-1 at 298 K in pyridine-d5). Of the two isomers the equatorial NH product was more stable than the axial one due to reduced 1,3 diaxial interactions. As a result, all NH axial product was converted to the equatorial isomer during subsequent steps in the synthesis. NMR and ab initio experiments confirmed the North-East structure of the 1',2'-azetidine locked nucleoside and North conformation of aza-ENA-T locked nucleosides with a chair conformation of the piperidine ring. The amino modified nucleosides were incorporated into different positions of a 15mer oligonucleotide. The azetidine modified AONs did not form stable duplexes with complementary RNA (ΔTm ~-1 to -4 °C), but they performed better than previously synthesized isosequential 1',2'-oxetane modified oligonucleotides. The 2',4'-aza-ENA-T modified oligonucleotide, on the other hand, showed excellent target affinity with complementary RNA (ΔTm ~+4 °C). The azetidine and aza-ENA-T modified oligonucleotides showed significant stability in the presence of human serum and snake venom phosphodiesterase (3'-exonuclease) as compared to the unmodified native sequence. The singly modified 15mer oligonucleotides were also subjected to RNase H promoted digestion in order to evaluate their potential as effective antisense agents. The effective enzyme activity (kcat/Km) was found to be lower in the modified AONs due to reduced enzyme-substrate binding. However, the catalytic activity of RNase H with these modified-AON:RNA duplexes were higher than observed with the native duplex.

Σκοπός: Να διερευνηθεί η επίδραση της καθημερινής χορήγησης σιλδεναφίλης στα επίπεδα διαλυτών δεικτών της ενδοθηλιακής λειτουργίας σε άνδρες με στυτική δυσλειτουργία. Μέθοδοι: Ασθενείς πάνω από 18 ετών με στυτική δυσλειτουργία αγγειακής αιτιολογίας για πάνω από 6 μήνες, είτε μόνη είτε σε συνδυασμό με νοσολογικές καταστάσεις ισχυρά συσχετιζόμενες με ενδοθηλιακή δυσλειτουργία όπως σακχαρώδης διαβήτης/μεταβολικό σύνδρομο, υπέρταση και στεφανιαία νόσος, έλαβαν σιλδεναφίλη 25 mg ημερησίως από του στόματος για 4 εβδομάδες. Δείκτες της ενδοθηλιακής λειτουργίας μετρήθηκαν στο πλάσμα στην αρχή και το τέλος της θεραπείας χρησιμοποιώντας τυπικές μεθόδους και εμπορικά διαθέσιμα υλικά. Αποτελέσματα: 112 άνδρες με μέση ηλικία (SD) 60,6 (7,3) έτη ολοκλήρωσαν το θεραπευτικό πρωτόκολλο. Η χορήγηση 25 mg σιλδεναφίλης καθημερινά για 4 εβδομάδες μείωσε σημαντικά τα επίπεδα της ενδοθηλίνης-1 σε σύγκριση με την αρχή της θεραπείας (2,83 ± 1,63 έναντι 3,24 ± 1,90 pg/ml, p<0,001). Σημαντικές αλλαγές παρατηρήθηκαν επίσης για το οξείδιο του αζώτου (ΝΟ) (35,12 ± 21,14 έναντι 31,91 ± 16,28 pmol/lt, p=0,01), τα επίπεδα της cGMP (3,79 ± 2,37 έναντι 2,70 ± 1.34 pmol/ml, p<0,001) και τον παράγοντα von Willebrand (956,08 ± 514,25 έναντι 1007,42 ± 466,25 mU/ml) αλλά όχι και για τους άλλους δείκτες που μετρήθηκαν (θρομβομοδουλίνη και Ε-σελεκτίνη). Η στυτική λειτουργία βελτιώθηκε επίσης. Συμπεράσματα: Η σιλδεναφίλη σε καθημερινή χορήγηση βελτιώνει την ενδοθηλιακή λειτουργία όπως αυτή εκτιμάται με τα επίπεδα βιολογικών δεικτών σε ασθενείς με στυτική δυσλειτουργία. Αυτά τα αποτελέσματα συμφωνούν με άλλες μελέτες που δείχνουν όμοια αποτελέσματα με θεραπεία με αναστολείς της φωσφοδιεστεράσης 5. Η κλινική σημασία των αποτελεσμάτων αυτών χρήζει περαιτέρω διερεύνησης. / Objective: To investigate the impact of daily sildenafil on levels of soluble molecular markers of endothelial function in men with erectile dysfunction. Methods: Patients over 18 years of age with erectile dysfunction of vascular aetiology for more than 6 months, either alone or in combination with disease states strongly associated with endothelial dysfunction such as diabetes/metabolic syndrome, hypertension and coronary artery disease, received sildenafil 25 mg orally for 4 weeks. Markers of endothelial function were measured in plasma at baseline and end-of-treatment using standard methods and commercially available kits. Results: 112 men with mean (SD) age of 60.6 (7.3) years completed the protocol. Sildenafil 25mg daily for 4 weeks significantly reduced endothelin-1 levels compared to baseline (2.83 ± 1.63 vs. 3.24 ± 1.90 pg/ml, p<0.001). Significant changes were also observed for nitric oxide (35.12 ± 21.14 vs. 31.91 ± 16.28 pmol/lt, p=0.01) and cyclic guanosine monophosphate (3.79 ± 2.37 vs. 2.70 ± 1.34 pmol/ml, p<0.001) and von Willebrand factor (956.08 ± 514.25 vs. 1007.42 ± 466.25 mU/ml) levels but not for the other biomarkers measured (thrombomodulin and E-selectin). Erectile function was significantly improved. Conclusions: Daily sildenafil improves endothelial function as assessed by levels of biomarkers of endothelial function in patients with erectile dysfunction. This is in agreement with other studies showing similar benefits with phosphodiesterase 5 inhibitor treatment. The clinical implications of this finding need further investigation.

L’hypertension systolique isolée (HSI) est le résultat de changements au niveau de la paroi vasculaire qui ont pour conséquence d’augmenter la rigidité artérielle. Ces modifications surviennent surtout au niveau des grosses artères comme l’aorte et sont associées au vieillissement. La fragmentation des fibres élastiques, leur calcification (élastocalcinose) et la fibrose font partie des changements majeurs observés avec l’âge. En plus de ces changements, le vieillissement vasculaire provoque des modifications au niveau des cellules qui composent la paroi. Les cellules endothéliales sécrètent moins de monoxyde d’azote (NO) provoquant une dysfonction endothéliale et les cellules musculaires lisses vasculaires (CMLVs) synthétisent maintenant des protéines matricielles et osseuses. Situé entre le sang et les CMLVs, l’endothélium contrôle le tonus vasculaire par la sécrétion de plusieurs substances vasoactives qui interagissent entre elles afin de maintenir l’homéostasie du système vasculaire. Parmi celles-ci, on note l’endothéline (ET), un puissant vasoconstricteur et le NO, un gaz vasorelaxants. Ce dernier est aussi reconnu pour bloquer la production d’ET par un mécanisme dépendant du guanosine monophosphate cyclique (GMPc). Comme il y a une interaction entre le NO et l’ET, et que cette dernière est impliquée dans la calcification artérielle, le NO pourrait être impliqué dans la modulation de l’élastocalcinose et de la rigidité artérielle par l’inhibition de l’ET et la modification de la composition de la paroi. Cet effet, qui se produirait au delà des effets vasorelaxants du NO, offre un potentiel thérapeutique intéressant pour l’HSI. Afin d’évaluer l’implication du NO dans la calcification vasculaire et la rigidité artérielle, un modèle animal d’HSI a été utilisé (modèle warfarine vitamine K, WVK). Ce modèle d’élastocalcinose est basé sur l’inhibition de la maturation d’une protéine anti-calcifiante, la matrix Gla protein (MGP), par la warfarine. Afin de déterminer l’implication physiologique du NO dans l’initiation et la progression de l’élastocalcinose, sa production a été inhibée par un analogue de la L-arginine, le L-NG-nitroarginine methyl ester (L-NAME). Lors des processus d’initiation de la calcification, le L-NAME a prévenu l’élastocalcinose sans toutefois modifier la vitesse de l’onde de pouls (PWV). Suite au traitement L-NAME, l’expression de la NO synthase inductible (iNOS) a été diminuée alors qu’elle a été augmentée lors du traitement WVK. Elle pourrait donc être impliquée dans les processus de calcification vasculaire. De plus, la NO synthase endothéliale (eNOS) semble également impliquée puisqu’elle a été augmentée dans le modèle WVK. Cette hausse pourrait être bénéfique pour limiter l’élastocalcinose alors que l’expression de la iNOS serait délétère. Lors de la progression de la calcification, le L-NAME a augmenté l’élastocalcinose et le PWV. Dans ce contexte, l’ET serait impliquée dans l’amplification de la calcification vasculaire entrainant une hausse de la rigidité artérielle. Comme le NO endogène limite la progression de la calcification et conséquemment la rigidité artérielle, il semble être protecteur. L’efficacité d’une modulation de la voie du NO dans le modèle WVK a été étudiée par l’administration d’un donneur de NO, le sinitrodil, ou d’un inhibiteur de la phosphosdiestérase 5 (PDE5), le tadalafil. La modulation de la voie du NO semble être bénéfique sur la rigidité artérielle, mais seulement de façon aiguë. En effet, le sinitrodil a modifié de transitoirement la rigidité au niveau de l’aorte possiblement par la modulation du tonus vasculaire sans toutefois avoir des effets sur la composition de la paroi. Comme le modèle WVK n’affecte pas la fonction endothéliale, les concentrations endogènes de NO semblent être optimales puisque le sinitrodil provoque une augmentation de l’élastocalcinose possiblement par le développement d’une tolérance. Tout comme le sinitrodil, le tadalafil a modulé de manière aiguë la rigidité artérielle sans modifier la composition de la paroi. Globalement, ces travaux ont permis de mettre en évidence les effets bénéfiques du NO endogène pour limiter le développement de l’HSI, suggérant qu’une dysfonction endothéliale, tel qu’observé lors du vieillissement, a un impact négatif sur la maladie. / Isolated systolic hypertension (ISH) is the result of complex changes in the vascular wall and consequently the increase of arterial stiffness. These modifications occur mainly in conductance arteries, like the aorta, and are associated with aging. The fragmentation of elastic fibers, calcification (elastocalcinosis), and fibrosis are major changes with age. In addition to these changes in the extracellular matrix, vascular aging also induces vascular cell wall modifications. These include decreased production of nitric oxide (NO) by endothelial cells, which induces endothelial dysfunction, and the production of matrix and bone proteins by vascular smooth muscle cells (VSMCs). Located between the blood and VSMCs, the endothelium controls vascular tone by secreting various vasoactive factors. These factors interact with each other to maintain the hemodynamic of the vascular system. Among these factors, the vasoconstrictor endothelin (ET) and the vasodilator NO. The latter has been shown to block ET production via a cyclic guanosine monophosphates-(cGMP) dependent mechanism, whereas ET has been implicated in arterial calcification. Therefore, NO might be involved in the modulation of elastocalcinosis and arterial stiffness by inhibiting ET and modifying the vascular wall composition. This effect of NO could offer interesting therapeutic potential for ISH. To evaluate the implication of NO in the vascular calcification and arterial stiffness, an animal model of ISH was used. This model of elastocalcinosis is based on the inhibition of the maturation of the anti-calcific protein, matrix Gla protein (MGP), by warfarin (WVK model). To gain insight into the physiological role of endogenous NO in the initiation and progression of elastocalcinosis, its production was inhibited by the administration of L-NAME. Interestingly, elastocalcinosis was prevented by L-NG-nitroarginine methyl ester (L-NAME) administration without any modifications of the pulse wave velocity (PWV) during the initiation of the calcification processes. After the L-NAME treatment, the expression of inducible NO synthase (iNOS) was decreased, whereas upon treatment with warfarin alone the expression of iNOS was increased, which could be implicated in vascular calcification and arterial stiffness. In addition, endothelial NO synthase (eNOS) seems to be implicated in this process as its expression was also increased upon WVK treatment. This increase could be beneficial to limit elastocalcinosis, whereas the increase in iNOS expression could be harmful. L-NAME administration during the progression of calcification increased elastocalcinosis and PWV. In an endothelial dysfunction context, ET has been shown to be involved in the amplification process of vascular calcification causing an increase in arterial stiffness. As NO limits the progression of calcification and consequently arterial stiffness, endogenous NO seems to be protective in the aorta. The efficacy of exogenous modulation of the NO pathway in the WVK model was studied upon administration of the NO donor, sinitrodil, or the phosphodiesterase type 5 inhibitor (PDE5), tadalafil. The exogenous modulation of the NO pathway seemed to be beneficial for arterial stiffness, but only in an acute manner. Indeed, sinitrodil modified the acute stiffness in the aorta potentially by vascular tone modulation, without having any effect on vascular wall composition. Since endothelial function was not affected upon WVK model, endogenous NO concentrations seem to be optimal. Thus, exogenous NO potentially caused an increase of elastocalcinosis by inducing tolerance to NO. As well as sinitrodil, tadalafil modulated the arterial stiffness in an acute manner without modifying the composition of the vascular wall. Broadly, these studies provide evidence that endogenous NO can limit ISH development, suggesting that endothelial dysfunction, as observed in aging, has a negative impact on this pathology.

Human genetic data suggests DISC1 (Disrupted-in-schizophrenia 1) is a susceptibility gene for schizophrenia and depression. Disc1 Q31L-/- mutants show depression-like behaviour and Disc1 L100P-/- mutants schizophrenia-like behaviour. Heterozygous mutants show an intermediate phenotype. In a gene-environment interaction study, we exposed heterozygotes to chronic social defeat (CSD) stress and phenotyped behaviour. Disc1, Bdnf(III) and Pde4b mRNA levels were also measured. Moreover, as epigenetic mechanisms may mediate some effects of CSD, we also exposed wildtype mice to CSD concurrently with the histone deacetylase inhibitor valproate. We found that CSD increased anxiety in L100P-/+ mutants, and that levels of Disc1, Bdnf(III) and Pde4b mRNA were higher in this mutant. Valproate treatment did not correct CSD-induced behavioural changes. In conclusion, we have demonstrated an interaction between a strong susceptibility gene for psychiatric disease and an environmental manipulation similar to stressors known to affect mental illness.

Human genetic data suggests DISC1 (Disrupted-in-schizophrenia 1) is a susceptibility gene for schizophrenia and depression. Disc1 Q31L-/- mutants show depression-like behaviour and Disc1 L100P-/- mutants schizophrenia-like behaviour. Heterozygous mutants show an intermediate phenotype. In a gene-environment interaction study, we exposed heterozygotes to chronic social defeat (CSD) stress and phenotyped behaviour. Disc1, Bdnf(III) and Pde4b mRNA levels were also measured. Moreover, as epigenetic mechanisms may mediate some effects of CSD, we also exposed wildtype mice to CSD concurrently with the histone deacetylase inhibitor valproate. We found that CSD increased anxiety in L100P-/+ mutants, and that levels of Disc1, Bdnf(III) and Pde4b mRNA were higher in this mutant. Valproate treatment did not correct CSD-induced behavioural changes. In conclusion, we have demonstrated an interaction between a strong susceptibility gene for psychiatric disease and an environmental manipulation similar to stressors known to affect mental illness.