This thesis aims to investigate fluoxetine, a widely prescribed SSRI antidepressant, for its role in the pathogenesis of NAFLD and uncover novel mechanisms by which it may contribute to drug-induced steatosis. We demonstrated that increased hepatic lipid accumulation was mediated, in part, via elevated serotonin production. The inhibition of hepatic serotonin synthesis prevented lipid accumulation in fluoxetine-treated hepatocytes demonstrating a causal role for serotonin in fluoxetine-induced hepatic steatosis. Interestingly, in several studies, serotonin signaling has been shown to impact prostaglandin biosynthesis. As prostaglandins have been implicated in the development of NAFLD, and fluoxetine has previously been shown to alter the production of prostaglandins I assessed the role of prostaglandins in fluoxetine-induced hepatic lipid accumulation. Fluoxetine treatment increased mRNA expression of prostaglandin biosynthetic enzymes, increased production of prostaglandin 15-deoxy-Δ12,14PGJ2 (PPARG agonist), and elevated PPARG targets involved in fatty acid uptake. Fluoxetine-induced lipid accumulation, 15-deoxy-Δ12,14PGJ2 production, and the expression of PPARG lipogenic genes were attenuated with a PTGS1 specific inhibitor. Taken together these findings suggested that fluoxetine-induced lipid accumulation was mediated via PTGS1 and its downstream product 15-deoxy-Δ12,14PGJ2. Given that Pparg was elevated following fluoxetine treatment, and PPARG regulates microRNA involved in hepatic lipid accumulation, my final project focused on PPARG’s role in altered miRNA expression. Indeed, fluoxetine treatment increased the miRNA expression of miR-122, an effect that was attenuated when fluoxetine treatment was combined with the PPARG antagonist GW9662, suggesting a fluoxetine-PPARG-miR122 axis contributing to hepatic steatosis. While these studies have only been performed in vitro, an understanding of the molecular changes associated with SSRI treatment may lead to the development of strategies to prevent the increased risk of adverse metabolic outcomes associated with the use of SSRI antidepressants. / Dissertation / Doctor of Philosophy (Medical Science) / In adults, major depressive disorder (depression) is one of the most common psychiatric illnesses. Recent data suggests that there are more than 4.1 million Canadians who currently suffer from depression. Depression is commonly treated using selective serotonin reuptake inhibitor (SSRI) antidepressants. While these antidepressants do help manage depressive symptoms, they can also cause unwanted side effects including a build-up of fat in the liver, leading to fatty liver disease. The goal of my research is to understand the link between SSRI use and the development of fatty liver disease. This thesis investigates the effects of fluoxetine (Prozac®), a commonly used SSRI antidepressant, on molecular pathways that can lead to the development of fatty liver disease. An understanding of the molecular changes with SSRI treatment may lead to the development of strategies to prevent the harmful effects of SSRI antidepressants on the liver.

La prostaglandine E2 est une hormone lipidique produite abondamment dans le corps, incluant dans le rein où elle agit localement pour réguler les fonctions rénales. Un couplage à la protéine Gαs menant à une production d’AMPc a classiquement été attribué au récepteur EP4 de PGE2. La signalisation d’EP4 s’est cependant avérée plus complexe et implique aussi un couplage aux protéines sensibles à la PTX Gαi et des effets reliés aux β-arrestines. Il y a maintenant plusieurs exemples de l’activation sélective de voies de signalisation indépendantes par des ligands des récepteurs couplés aux protéines G (RCPG), et ce concept désigné sélectivité fonctionnelle pourrait être exploité dans le développement de nouveaux médicaments plus spécifiques et efficaces. Dans une première étude, la puissance et l’activité intrinsèque d’une série de ligands d’EP4 pour l’activation de Gαs, Gαi et de la ß-arrestine ont été systématiquement déterminées relativement au ligand endogène PGE2. Dans ce but, trois essais de transfert d’énergie de résonance de bioluminescence (BRET) ont été adaptés pour évaluer les différentes voies dans des cellules vivantes. Nos résultats montrent une sélectivité fonctionnelle importante parmi les agonistes évalués et ont une implication pour l’utilisation d’analogues de la PGE2 dans un contexte expérimental et possiblement clinique, puisque leur spectre d’activité diffère de l’agoniste naturel. La méthodologie basée sur le BRET utilisée lors de cette première évaluation systématique d’une série d’agonistes d’EP4 devrait être applicable à l’étude d’autres RCPG. Dans une deuxième étude, des peptides reproduisant des régions juxtamembranaires extracellulaires du récepteur EP4 ont été conçus selon le raisonnement que des peptides ciblant des régions éloignées du site de liaison du ligand naturel ont le potentiel de ne moduler qu’une partie des activités du récepteur. L’insuffisance rénale aiguë est une complication médicale grave caractérisée par un déclin brusque et soutenu de la fonction rénale et pour laquelle il n’y a pas de traitement efficace à l’heure actuelle. Nos résultats montrent que le peptidomimétique dérivé d’EP4 optimisé (THG213.29) améliore significativement les fonctions rénales et les changements histologiques dans une insuffisance rénale aiguë induite par cisplatine ou par occlusion des artères rénales dans des rats Sprague-Dawley. Le THG213.29 ne compétitionnait pas la liaison de la PGE2 à EP4, mais modulait la cinétique de dissociation de la PGE2, suggérant une liaison à un site allostérique d’EP4. Le THG213.29 démontrait une sélectivité fonctionnelle, puisqu’il inhibait partiellement la production d’AMPc induite par EP4 mais n’affectait pas l’activation de Gαi ou le recrutement de la ß-arrestine. Nos résultats indiquent que le THG213.29 représente une nouvelle classe d’agent diurétique possédant les propriétés d’un modulateur allostérique non-compétitif des fonctions du récepteur EP4 pour l’amélioration des fonctions rénales suite à une insuffisance rénale aiguë. / Prostaglandin E2 (PGE2) is a lipid hormone mediator widely produced in the body, including in the kidney where it acts locally to regulate renal function. Classically, the PGE2 receptor EP4 has been classified as coupling to the Gαs subunit, leading to intracellular cAMP increases. However EP4 signaling has been revealed to be more complex and also involves coupling to PTX-sensitive Gαi proteins and ß-arrestin mediated effects. There are now many examples of selective activation of independent pathways by G-protein coupled receptor (GPCR) ligands, a concept referred to as functional selectivity that could be exploited for the development of more specific and efficacious drugs. In a first study, the potencies and efficacies of a panel of EP4 ligands were systematically determined for the activation of Gαs, Gαi and ß-arrestin relative to the endogenous ligand PGE2. For this purpose, three bioluminescence resonance energy transfer (BRET) assays were adapted to evaluate the respective pathways in living cells. Our results suggest considerable functional selectivity among the tested, structurally related agonists and have implications for the use of PGE2 analogues in experimental and possibly clinical settings, as their activity spectra on EP4 differ from that of the native agonist. The BRET-based methodology used for this first systematic assessment of a set of EP4 agonists should be applicable for the study of other GPCRs. In a second study, peptides were derived from extracellular juxtamembranous regions of the EP4 receptor following the rationale that peptides that target regions of the receptor remote of the ligand-binding site might modulate a subset of the EP4-mediated activities. Acute renal failure is a serious medical complication characterized by an abrupt and sustained decline in renal function and for which there is currently no effective treatment. Our results show that the optimized EP4-derived peptidomimetic THG213.29 significantly improved renal functions and histological changes in acute renal failure induced by either cisplatin or renal artery occlusion in Sprague-Dawley rats. THG213.29 did not displace PGE2 binding to EP4, but modulated PGE2 binding dissociation kinetics, indicative of an allosteric binding mode. THG213.29 exhibited functional selectivity, as it partially inhibited EP4-mediated cAMP production but did not affect Gαi activation or ß-arrestin recruitment. Our results demonstrate that THG213.29 represents a novel class of diuretic agent with noncompetitive allosteric modulator effects on EP4 receptor function for improving renal function following acute renal failure.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal.

No description available.

La placentation humaine est de type hémomonochoriale, le sang maternel est directement en contact avec le syncytiotrophoblaste. Les flux sanguins maternels, dans la chambre intervilleuse, exercent des forces mécaniques de cisaillement (shear stress) sur la surface microvillositaire du syncytiotrophoblaste. Les effets physiologiques du shear stress exercé par les flux sanguins sur l’endothélium vasculaire artériel et veineux ont fait l’objet de nombreux travaux scientifiques. En revanche, les effets biologiques du shear stress sur le syncytiotrophoblaste humain n’ont jamais été explorés. L’objectif de ce travail était premièrement d’évaluer les valeurs du shear stress exercé in vivo sur le syncytiotrophoblaste humain au cours des grossesses normales, puis de mettre au point un modèle de culture primaire dynamique afin de reproduire les conditions physiologique et d’étudier in vitro la réponse biologique du syncytiotrophoblaste au shear stress. En dépit d’un débit sanguin maternel intraplacentaire important, estimé entre 400 et 600 mL.min-1, le shear stress moyen exercée par le syncytiotrophoblaste est estimée entre 0.5±0.2 et 2.3±1.1 dyn.cm-2. Nos résultats montrent cependant que l’intensité du shear stress est très hétérogène tant à l’échelle de la chambre intervilleuse que de la villosité terminale. Nous avons développé un modèle de culture cellulaire dynamique en condition de flux adapté au syncytiotrophoblaste humain. Ce modèle permet d’appliquer un shear stress égal et constant sur toutes les cellules cultivées et reproductible à chaque culture primaire. Aux gammes de shear stress étudiées (1 dyn.cm-2), nous n’avons pas mis en évidence de diminution de la viabilité cellulaire ni de déclenchement des processus précoces d’apoptose en conditions dynamiques comparativement aux conditions statiques. Deux types de chambre de perfusion permettent d’étudier des réponses cellulaires au shear stress à court et long terme selon des temps d’exposition allant de 5 minutes à 24 heures. Ce modèle expérimental a permis de montrer que le syncytiotrophoblaste humain en culture primaire est mécanosensible. La réponse cellulaire à des niveaux de shear stress de 1 dyn.cm-2 est multiple selon les temps d’exposition et le niveau d’intégration étudié. Après 45 minutes de shear stress les taux d’AMP cyclique intracellulaires sont augmentés ce qui a pour effet d’activer la voie de signalisation intracellulaire PKA-CREB. Cette augmentation d’AMP cyclique est secondaire à la synthèse et la libération de prostaglandine E2 qui, par une boucle de régulation autocrine stimule l’adenylate cyclase. L’augmentation de la synthèse/libération de PGE2 est dépendante de l’augmentation rapide du calcium intracellulaire sous shear stress. L’exposition au shear stress de 24 heures stimule l’expression et la sécrétion du PlGF, un facteur de croissance indispensable à l’angiogenèse placentaire et pour l’adaptation maternelle à la grossesse sur le plan vasculaire. Nos travaux montrent que l’augmentation de l’AMPc intracellulaire et l’activation de la PKA contribuent à la phosphorylation de CREB, facteur de transcription régulant l’expression du PlGF. / Human placentation is hemomonochorial, maternal blood circulates in direct contact with the syncytiotrophoblast. In the intervillous space, the maternal blood exerts frictional mechanical forces (shear stress) on the microvillous surface of the syncytiotrophoblast. Flowing blood constantly exerts a shear stress, on the endothelial cells lining blood vessel walls, and the endothelial cells respond to shear stress by changing their morphology, function, and gene expression. The effects of shear stress on the human syncytiotrophoblast and its biological functions have never been studied. The objectives of this study were (1) to determine in silico the physiological values of shear stress exerted on human syncytiotrophoblast during normal pregnancies, (2) to develop a model reproducing in vitro the shear stress on human syncytiotrophoblast and (3) to study in vitro the biological response of human syncytiotrophoblast to shear stress. The 2D numerical simulations showed that the shear stress applied to the syncytiotrophoblast is highly heterogeneous in the intervillous space. In spite of high intraplacental maternal blood flow rates (400-600mL.min-1), the estimated average values of shear stress are relatively low (0.5±0.2 to 2.3±1.1 dyn.cm-2). To study the shear stress-induced cellular responses during exposure times ranging from 5 minutes to 24 hours we have developed two dynamic cell culture models adapted to the human syncytiotrophoblast. We found no evidence of decreased cell viability or early processes of apoptosis in dynamic conditions (1 dyn.cm-2, 24h) compared to static conditions. Shear stress (1 dyn.cm-2) triggers intracellular calcium flux, which increases the synthesis and release of PGE2. The enhanced intracellular cAMP in FSS conditions was blocked by COX1/COX2 inhibitors, suggesting that the increase in PGE2 production could activate the cAMP/PKA pathway in an autocrine/paracrine fashion. FSS activates the cAMP/PKA pathway leading to upregulation of PlGF in human STB. Shear stress-induced phosphorylation of CREB and upregulation of PlGF were prevented by inhibition of PKA with H89 (3 μM). The syncytiotrophoblast of the human placenta is a mechanosenstive tissue.

Impaired endothelial vasomotor function is a hallmark of many chronic disease states, including essential hypertension and type 2 diabetes mellitus. Loss of the homeostatic role of the endothelium in large conduit arteries can contribute to the pathogenesis of cardiovascular conditions in these vessels (e.g. stroke, atherosclerosis). A fundamental understanding of mechanisms controlling endothelial function in hypertension and type 2 diabetes mellitus is required for appropriate clinical strategies targeting the cardiovascular conditions associated with these diseases. The vast majority of basic science studies examining endothelial function in animal models of hypertension and type 2 diabetes have been conducted in males. Studying endothelial function in females is imperative for determining potential sex-specific mechanisms of dysfunction and thus appropriate therapeutic strategies. Thus the global purpose of this thesis is to identify and characterize the pathways controlling impaired vasomotor function in female animal models of two chronic disease states: hypertension and type 2 diabetes mellitus. Chapters 2 and 3 of this thesis examine sex differences in endothelium-dependent vasorelaxation (EDR) and vasocontraction (EDC) of aortic segments isolated from male and female spontaneously hypertensive rats (SHR), a model of essential hypertension, as the animals age between 16 and 30 wk old. All endothelial vasomotor data presented in the Abstract are peak responses to 10⁻⁵ M acetylcholine. Endothelial vasomotor impairment is represented by lower EDR or by higher EDC. These present data confirmed well-established findings from the literature that 16 wk old male SHR exhibit endothelial vasomotor impairments (EDR: 77±4 %; EDC: 76±7 %) compared to normotensive Wistar-Kyoto (WKY; EDR: 89±6 %; EDC: 59±8 %; p<0.05) controls, and that this impairment worsens with ageing in 30 wk male SHR (EDR: 63±2 %; EDC: 91±3 %; p<0.05). The observation that EDR was reduced in 30 wk female SHR (EDR: 76±4 %) compared to 16 wk counterparts (EDR: 101±2 %; p<0.05), however, was novel and interesting, as there were previously no reports of vasomotor responses in female SHR older than 19 wk. Moreover, the blunted EDR response of 30 wk female SHR approached the level of impairment exhibited by 30 wk male SHR (but was still slightly greater in females; p<0.05). The limited sex difference of the EDR within 30 wk SHR (males –13 % vs. females; p<0.05) contrasted that of 16 wk SHR (males –24 % vs. females; p<0.05), when the robust and unimpaired relaxation displayed by females was much greater than the significantly blunted response of males. Interestingly, endothelium-dependent contractions in quiescent rings were moderate and similar between 16 wk (EDC: 50±4 %) and 30 wk female SHR (EDC: 59±7 %; p=N/S) as compared to the greater contractions of males that were exacerbated with ageing (see above; p<0.05 both sex and ageing comparison). A major role has been established for the cyclooxygenase (COX)-1-thromboxane A₂/prostaglandin (TP) receptor pathway in the impaired endothelial vasomotor function of male SHR. Indeed, a similar mechanism appears to be responsible for the dysfunction observed in 30 wk female SHR in this thesis since robust endothelial function was restored in these animals with both antagonism of TP receptor (EDR: 111±2 %; EDC: 7±2 %; p<0.05) and preferential inhibition of COX-1 (EDR: 112±3 %; EDC: –5±3 %; p<0.05). In contrast, preferential inhibition of COX-2 only partially tempered endothelial impairments of 30 wk female SHR (EDR: 99±5 %; EDC: 27±3 %; p<0.05), suggesting that, similar to ageing male SHR, this isoform makes at most a secondary contribution to the dysfunction in 30 wk female SHR. Collectively, these data indicate that ageing female SHR exhibit a mechanism of endothelial impairment that is similar to that of male SHR and that is largely COX-1- and TP receptor-dependent. Chapter 4 examines the ability of chronic dietary administration of the n-3 polyunsaturated fatty acid (PUFA), docosahexaenoic acid (DHA, 22:6 n-3), to ameliorate endothelial vasomotor function in adult male SHR with established hypertension. The impaired endothelial function of aortic segments isolated from adult male SHR (EDR: 48±6 %) was not improved following 10–12 wk of DHA feeding (EDR: 45±5 %; p=N/S). This finding was unexpected since it has been shown in the literature that feeding other n-3 PUFAs improves vasomotor responses in younger SHR, in which hypertension and its associated consequences are still developing. This is the first report of the effects of n-3 PUFA on endothelial vasomotor responses in adult SHR with established hypertension. These data suggest that dietary DHA do not improve vasomotor function in adult SHR. Chapter 5 examines α₁ adrenergic contraction and EDR of aortic segments isolated from 14 wk old female Zucker diabetic fatty rats (ZDF), a genetic model of high fat diet-induced obesity and type 2 diabetes, and lean non-diabetic female Zucker Lean rats. Additionally, some ZDF received an 8 wk administration of anti-diabetic metformin drug therapy, aerobic exercise training, or a combination of the two. Maximal α₁ adrenergic contractions were over 2-fold higher in high fat-fed ZDF (1.69±0.16 g) compared to Lean (0.71±0.13 g; p<0.05). This elevation in ZDF was abolished by exercise training alone (1.02±0.17 g; p<0.05) but was not altered by metformin (1.56±0.19 g; p=N/S). In contrast to the severely impaired endothelial vasomotor function reported in male ZDF in the literature, robust EDR was observed in female ZDF (72±7 %) that was similar to Lean (75±6 %; p=N/S) and that was unaltered by exercise training (76±5 %; p=N/S) or metformin (76±6 %; p=N/S). These results indicate that enhanced α₁ adrenergic contraction is a mechanism of altered vasomotor function in female type 2 diabetic ZDF rats and that it could possibly be addressed by a chronic exercise training intervention. The main novelty of the thesis is the extension of the current understanding of endothelial vasomotor function to hypertensive and type 2 diabetic females. The knowledge gained from examining mechanisms involved in endothelial impairments in ageing hypertensive females and from testing the therapeutic potential of currently used anti-diabetic interventions in the type 2 diabetic female vasculature has interesting potential application. This basic scientific information could help direct clinical therapeutic strategies to target population-specific mechanisms of dysfunction. Understanding female sex-specific endothelial behaviour in patient populations is important for describing cardiovascular complications, defining mechanisms, and applying appropriate therapeutic targets. Findings from this thesis indicate a sex-dependence of the total divergence of endothelial function (e.g. female type 2 diabetic rats vs. male counterparts in the literature) and of the interaction of disease variables (e.g. age) and endothelial vasomotor responses.

Impaired endothelial vasomotor function is a hallmark of many chronic disease states, including essential hypertension and type 2 diabetes mellitus. Loss of the homeostatic role of the endothelium in large conduit arteries can contribute to the pathogenesis of cardiovascular conditions in these vessels (e.g. stroke, atherosclerosis). A fundamental understanding of mechanisms controlling endothelial function in hypertension and type 2 diabetes mellitus is required for appropriate clinical strategies targeting the cardiovascular conditions associated with these diseases. The vast majority of basic science studies examining endothelial function in animal models of hypertension and type 2 diabetes have been conducted in males. Studying endothelial function in females is imperative for determining potential sex-specific mechanisms of dysfunction and thus appropriate therapeutic strategies. Thus the global purpose of this thesis is to identify and characterize the pathways controlling impaired vasomotor function in female animal models of two chronic disease states: hypertension and type 2 diabetes mellitus. Chapters 2 and 3 of this thesis examine sex differences in endothelium-dependent vasorelaxation (EDR) and vasocontraction (EDC) of aortic segments isolated from male and female spontaneously hypertensive rats (SHR), a model of essential hypertension, as the animals age between 16 and 30 wk old. All endothelial vasomotor data presented in the Abstract are peak responses to 10⁻⁵ M acetylcholine. Endothelial vasomotor impairment is represented by lower EDR or by higher EDC. These present data confirmed well-established findings from the literature that 16 wk old male SHR exhibit endothelial vasomotor impairments (EDR: 77±4 %; EDC: 76±7 %) compared to normotensive Wistar-Kyoto (WKY; EDR: 89±6 %; EDC: 59±8 %; p<0.05) controls, and that this impairment worsens with ageing in 30 wk male SHR (EDR: 63±2 %; EDC: 91±3 %; p<0.05). The observation that EDR was reduced in 30 wk female SHR (EDR: 76±4 %) compared to 16 wk counterparts (EDR: 101±2 %; p<0.05), however, was novel and interesting, as there were previously no reports of vasomotor responses in female SHR older than 19 wk. Moreover, the blunted EDR response of 30 wk female SHR approached the level of impairment exhibited by 30 wk male SHR (but was still slightly greater in females; p<0.05). The limited sex difference of the EDR within 30 wk SHR (males –13 % vs. females; p<0.05) contrasted that of 16 wk SHR (males –24 % vs. females; p<0.05), when the robust and unimpaired relaxation displayed by females was much greater than the significantly blunted response of males. Interestingly, endothelium-dependent contractions in quiescent rings were moderate and similar between 16 wk (EDC: 50±4 %) and 30 wk female SHR (EDC: 59±7 %; p=N/S) as compared to the greater contractions of males that were exacerbated with ageing (see above; p<0.05 both sex and ageing comparison). A major role has been established for the cyclooxygenase (COX)-1-thromboxane A₂/prostaglandin (TP) receptor pathway in the impaired endothelial vasomotor function of male SHR. Indeed, a similar mechanism appears to be responsible for the dysfunction observed in 30 wk female SHR in this thesis since robust endothelial function was restored in these animals with both antagonism of TP receptor (EDR: 111±2 %; EDC: 7±2 %; p<0.05) and preferential inhibition of COX-1 (EDR: 112±3 %; EDC: –5±3 %; p<0.05). In contrast, preferential inhibition of COX-2 only partially tempered endothelial impairments of 30 wk female SHR (EDR: 99±5 %; EDC: 27±3 %; p<0.05), suggesting that, similar to ageing male SHR, this isoform makes at most a secondary contribution to the dysfunction in 30 wk female SHR. Collectively, these data indicate that ageing female SHR exhibit a mechanism of endothelial impairment that is similar to that of male SHR and that is largely COX-1- and TP receptor-dependent. Chapter 4 examines the ability of chronic dietary administration of the n-3 polyunsaturated fatty acid (PUFA), docosahexaenoic acid (DHA, 22:6 n-3), to ameliorate endothelial vasomotor function in adult male SHR with established hypertension. The impaired endothelial function of aortic segments isolated from adult male SHR (EDR: 48±6 %) was not improved following 10–12 wk of DHA feeding (EDR: 45±5 %; p=N/S). This finding was unexpected since it has been shown in the literature that feeding other n-3 PUFAs improves vasomotor responses in younger SHR, in which hypertension and its associated consequences are still developing. This is the first report of the effects of n-3 PUFA on endothelial vasomotor responses in adult SHR with established hypertension. These data suggest that dietary DHA do not improve vasomotor function in adult SHR. Chapter 5 examines α₁ adrenergic contraction and EDR of aortic segments isolated from 14 wk old female Zucker diabetic fatty rats (ZDF), a genetic model of high fat diet-induced obesity and type 2 diabetes, and lean non-diabetic female Zucker Lean rats. Additionally, some ZDF received an 8 wk administration of anti-diabetic metformin drug therapy, aerobic exercise training, or a combination of the two. Maximal α₁ adrenergic contractions were over 2-fold higher in high fat-fed ZDF (1.69±0.16 g) compared to Lean (0.71±0.13 g; p<0.05). This elevation in ZDF was abolished by exercise training alone (1.02±0.17 g; p<0.05) but was not altered by metformin (1.56±0.19 g; p=N/S). In contrast to the severely impaired endothelial vasomotor function reported in male ZDF in the literature, robust EDR was observed in female ZDF (72±7 %) that was similar to Lean (75±6 %; p=N/S) and that was unaltered by exercise training (76±5 %; p=N/S) or metformin (76±6 %; p=N/S). These results indicate that enhanced α₁ adrenergic contraction is a mechanism of altered vasomotor function in female type 2 diabetic ZDF rats and that it could possibly be addressed by a chronic exercise training intervention. The main novelty of the thesis is the extension of the current understanding of endothelial vasomotor function to hypertensive and type 2 diabetic females. The knowledge gained from examining mechanisms involved in endothelial impairments in ageing hypertensive females and from testing the therapeutic potential of currently used anti-diabetic interventions in the type 2 diabetic female vasculature has interesting potential application. This basic scientific information could help direct clinical therapeutic strategies to target population-specific mechanisms of dysfunction. Understanding female sex-specific endothelial behaviour in patient populations is important for describing cardiovascular complications, defining mechanisms, and applying appropriate therapeutic targets. Findings from this thesis indicate a sex-dependence of the total divergence of endothelial function (e.g. female type 2 diabetic rats vs. male counterparts in the literature) and of the interaction of disease variables (e.g. age) and endothelial vasomotor responses.

The expanding therapeutic use of thalidomide (TD) remains limited by its species-specific teratogenicity in humans and rabbits, but not rodents. The R and S isomers of TD may be selectively responsible for its respective therapeutic and teratogenic effects, but rapid in vivo racemization makes this impossible to confirm. Fluorothalidomide (FTD), a fluorinated TD analogue with stable, non-racemizing isomers, may serve as a model compound for determining stereoselective effects. In vivo, FTD was undetectable in plasma, suggesting rapid breakdown, as confirmed in vitro, where FTD hydrolyzed up to 22-fold faster than TD. Unlike TD, FTD in pregnant rabbits and mice was highly toxic and lethal to both dams and fetuses. In rabbit embryo culture, FTD initiated optic (eye) vesicle and hindbrain but not classic limb bud embryopathies. Chemical instability, potent general toxicity and absence of limb bud embryopathies make FTD an unsuitable stereoselective model for TD teratogenesis. TD teratogenesis may involve its bioactivation by embryonic prostaglandin H synthases (PHSs) to a free radical intermediate that increases embryopathic reactive oxygen species (ROS) formation. However, the teratogenic potential of rapidly formed TD hydrolysis products and the determinants of species-specific teratogenesis are unclear. For some teratogens, mouse strains that are resistant in vivo are susceptible in embryo culture, suggesting maternal and/or placental determinants of risk. However, TD and two hydrolysis products, 2-phthalimidoglutaramic acid (PGMA) and 2-phthalimidoglutaraic acid (PGA), were non-embryopathic in CD-1 mouse embryo culture. Also, mice deficient in oxoguanine glycosylase 1 (OGG1), which repairs oxidatively damaged DNA, were resistant to TD embryopathies in culture and in vivo. Therefore, murine resistance to TD teratogenesis is dependent on embryonic factors, rather than maternal/placental determinants or increased DNA repair. In contrast, rabbit embryos exposed in culture to TD, PGMA and PGA exhibited head/brain, otic (ear) vesicle and classic limb bud embryopathies, validating the first mammalian embryo culture model for TD teratogenesis and providing the first evidence of a teratogenic role for TD hydrolysis products. Pretreatment with eicosatetraynoic acid (ETYA), a dual PHS/lipoxygenase inhibitor, or phenylbutylnitrone (PBN), a free radical spin trapping agent, completely blocked TD, PGMA and PGA-initiated embryopathies, implicating a PHS-dependent, ROS-mediated embryopathic mechanism.

The expanding therapeutic use of thalidomide (TD) remains limited by its species-specific teratogenicity in humans and rabbits, but not rodents. The R and S isomers of TD may be selectively responsible for its respective therapeutic and teratogenic effects, but rapid in vivo racemization makes this impossible to confirm. Fluorothalidomide (FTD), a fluorinated TD analogue with stable, non-racemizing isomers, may serve as a model compound for determining stereoselective effects. In vivo, FTD was undetectable in plasma, suggesting rapid breakdown, as confirmed in vitro, where FTD hydrolyzed up to 22-fold faster than TD. Unlike TD, FTD in pregnant rabbits and mice was highly toxic and lethal to both dams and fetuses. In rabbit embryo culture, FTD initiated optic (eye) vesicle and hindbrain but not classic limb bud embryopathies. Chemical instability, potent general toxicity and absence of limb bud embryopathies make FTD an unsuitable stereoselective model for TD teratogenesis. TD teratogenesis may involve its bioactivation by embryonic prostaglandin H synthases (PHSs) to a free radical intermediate that increases embryopathic reactive oxygen species (ROS) formation. However, the teratogenic potential of rapidly formed TD hydrolysis products and the determinants of species-specific teratogenesis are unclear. For some teratogens, mouse strains that are resistant in vivo are susceptible in embryo culture, suggesting maternal and/or placental determinants of risk. However, TD and two hydrolysis products, 2-phthalimidoglutaramic acid (PGMA) and 2-phthalimidoglutaraic acid (PGA), were non-embryopathic in CD-1 mouse embryo culture. Also, mice deficient in oxoguanine glycosylase 1 (OGG1), which repairs oxidatively damaged DNA, were resistant to TD embryopathies in culture and in vivo. Therefore, murine resistance to TD teratogenesis is dependent on embryonic factors, rather than maternal/placental determinants or increased DNA repair. In contrast, rabbit embryos exposed in culture to TD, PGMA and PGA exhibited head/brain, otic (ear) vesicle and classic limb bud embryopathies, validating the first mammalian embryo culture model for TD teratogenesis and providing the first evidence of a teratogenic role for TD hydrolysis products. Pretreatment with eicosatetraynoic acid (ETYA), a dual PHS/lipoxygenase inhibitor, or phenylbutylnitrone (PBN), a free radical spin trapping agent, completely blocked TD, PGMA and PGA-initiated embryopathies, implicating a PHS-dependent, ROS-mediated embryopathic mechanism.

La prostaglandine E2 est une hormone lipidique produite abondamment dans le corps, incluant dans le rein où elle agit localement pour réguler les fonctions rénales. Un couplage à la protéine Gαs menant à une production d’AMPc a classiquement été attribué au récepteur EP4 de PGE2. La signalisation d’EP4 s’est cependant avérée plus complexe et implique aussi un couplage aux protéines sensibles à la PTX Gαi et des effets reliés aux β-arrestines. Il y a maintenant plusieurs exemples de l’activation sélective de voies de signalisation indépendantes par des ligands des récepteurs couplés aux protéines G (RCPG), et ce concept désigné sélectivité fonctionnelle pourrait être exploité dans le développement de nouveaux médicaments plus spécifiques et efficaces. Dans une première étude, la puissance et l’activité intrinsèque d’une série de ligands d’EP4 pour l’activation de Gαs, Gαi et de la ß-arrestine ont été systématiquement déterminées relativement au ligand endogène PGE2. Dans ce but, trois essais de transfert d’énergie de résonance de bioluminescence (BRET) ont été adaptés pour évaluer les différentes voies dans des cellules vivantes. Nos résultats montrent une sélectivité fonctionnelle importante parmi les agonistes évalués et ont une implication pour l’utilisation d’analogues de la PGE2 dans un contexte expérimental et possiblement clinique, puisque leur spectre d’activité diffère de l’agoniste naturel. La méthodologie basée sur le BRET utilisée lors de cette première évaluation systématique d’une série d’agonistes d’EP4 devrait être applicable à l’étude d’autres RCPG. Dans une deuxième étude, des peptides reproduisant des régions juxtamembranaires extracellulaires du récepteur EP4 ont été conçus selon le raisonnement que des peptides ciblant des régions éloignées du site de liaison du ligand naturel ont le potentiel de ne moduler qu’une partie des activités du récepteur. L’insuffisance rénale aiguë est une complication médicale grave caractérisée par un déclin brusque et soutenu de la fonction rénale et pour laquelle il n’y a pas de traitement efficace à l’heure actuelle. Nos résultats montrent que le peptidomimétique dérivé d’EP4 optimisé (THG213.29) améliore significativement les fonctions rénales et les changements histologiques dans une insuffisance rénale aiguë induite par cisplatine ou par occlusion des artères rénales dans des rats Sprague-Dawley. Le THG213.29 ne compétitionnait pas la liaison de la PGE2 à EP4, mais modulait la cinétique de dissociation de la PGE2, suggérant une liaison à un site allostérique d’EP4. Le THG213.29 démontrait une sélectivité fonctionnelle, puisqu’il inhibait partiellement la production d’AMPc induite par EP4 mais n’affectait pas l’activation de Gαi ou le recrutement de la ß-arrestine. Nos résultats indiquent que le THG213.29 représente une nouvelle classe d’agent diurétique possédant les propriétés d’un modulateur allostérique non-compétitif des fonctions du récepteur EP4 pour l’amélioration des fonctions rénales suite à une insuffisance rénale aiguë. / Prostaglandin E2 (PGE2) is a lipid hormone mediator widely produced in the body, including in the kidney where it acts locally to regulate renal function. Classically, the PGE2 receptor EP4 has been classified as coupling to the Gαs subunit, leading to intracellular cAMP increases. However EP4 signaling has been revealed to be more complex and also involves coupling to PTX-sensitive Gαi proteins and ß-arrestin mediated effects. There are now many examples of selective activation of independent pathways by G-protein coupled receptor (GPCR) ligands, a concept referred to as functional selectivity that could be exploited for the development of more specific and efficacious drugs. In a first study, the potencies and efficacies of a panel of EP4 ligands were systematically determined for the activation of Gαs, Gαi and ß-arrestin relative to the endogenous ligand PGE2. For this purpose, three bioluminescence resonance energy transfer (BRET) assays were adapted to evaluate the respective pathways in living cells. Our results suggest considerable functional selectivity among the tested, structurally related agonists and have implications for the use of PGE2 analogues in experimental and possibly clinical settings, as their activity spectra on EP4 differ from that of the native agonist. The BRET-based methodology used for this first systematic assessment of a set of EP4 agonists should be applicable for the study of other GPCRs. In a second study, peptides were derived from extracellular juxtamembranous regions of the EP4 receptor following the rationale that peptides that target regions of the receptor remote of the ligand-binding site might modulate a subset of the EP4-mediated activities. Acute renal failure is a serious medical complication characterized by an abrupt and sustained decline in renal function and for which there is currently no effective treatment. Our results show that the optimized EP4-derived peptidomimetic THG213.29 significantly improved renal functions and histological changes in acute renal failure induced by either cisplatin or renal artery occlusion in Sprague-Dawley rats. THG213.29 did not displace PGE2 binding to EP4, but modulated PGE2 binding dissociation kinetics, indicative of an allosteric binding mode. THG213.29 exhibited functional selectivity, as it partially inhibited EP4-mediated cAMP production but did not affect Gαi activation or ß-arrestin recruitment. Our results demonstrate that THG213.29 represents a novel class of diuretic agent with noncompetitive allosteric modulator effects on EP4 receptor function for improving renal function following acute renal failure.