Use of diuretics and risk of incident gout: a population-based case-control study

OBJECTIVE Use of diuretics has been associated with an increased risk of gout. Data on different types of diuretics are scarce. We undertook this study to investigate the association between use of loop diuretics, thiazide or thiazide-like diuretics, and potassium-sparing agents and the risk of developing incident gout. METHODS We conducted a retrospective population-based case-control analysis using the General Practice Research Database established in the UK. We identified case patients who were diagnosed as having incident gout between 1990 and 2010. One control patient was matched to each case patient for age, sex, general practice, calendar time, and years of active history in the database. We used conditional logistic regression to calculate odds ratios (ORs) and 95% confidence intervals (95% CIs), and we adjusted for potential confounders. RESULTS We identified 91,530 incident cases of gout and the same number of matched controls. Compared to past use of diuretics from each respective drug class, adjusted ORs for current use of loop diuretics, thiazide diuretics, thiazide-like diuretics, and potassium-sparing diuretics were 2.64 (95% CI 2.47-2.83), 1.70 (95% CI 1.62-1.79), 2.30 (95% CI 1.95-2.70), and 1.06 (95% CI 0.91-1.23), respectively. Combined use of loop diuretics and thiazide diuretics was associated with the highest relative risk estimates of gout (adjusted OR 4.65 [95% CI 3.51-6.16]). Current use of calcium channel blockers or losartan slightly attenuated the risk of gout in patients who took diuretics. CONCLUSION Use of loop diuretics, thiazide diuretics, and thiazide-like diuretics was associated with an increased risk of incident gout, although use of potassium-sparing agents was not.

Taste acceptability of pulverized brand-name and generic drugs containing amlodipine or candesartan.

Trials with pulverized brand-name antihypertensive drugs suggest that, from the perspective of taste acceptability, crushed candesartan, chlortalidon, hydrochlorothiazide, lercanidipine and lisinopril should be preferred to pulverized amlodipine, atenolol, bisoprolol, enalapril, irbesartan, losartan, ramipril, telmisartan and valsartan. Brand-name antihypertensive drugs and the corresponding generic medicines have never been compared with respect to their taste acceptability. We therefore investigated among healthy health care workers the taste acceptability of a pulverized 1 mg-test dose of the brand-name and two generics containing either the dihydropyridine calcium-channel blocker amlodipine (Norvasc(®), Amlodipin-Mepha(®) and Amlodipin Pfizer(®)) or the angiotensin receptor antagonist candesartan (Atacand(®), Cansartan-Mepha(®) and Pemzek(®)). For this purpose, a smiley-face scale depicting four degrees of pleasure was used. Between November and December 2013, the taste test was performed among 19 nurses (15 female and 4 male subjects) and 12 physicians (5 female and 7 male subjects) aged between 25 and 49 years. Pulverized brand-names and generics containing either amlodipine or candesartan did not differ with respect to their taste acceptability.

The role of AT1 receptor autoantibodies in the pathophysiology of preeclampsia

Preeclampsia is a disease that affects 3–5% of all pregnancies. The cause is unknown and there is currently no treatment. The disease poses significant health risks to both the mother and the fetus. To date, research on the topic has not produced a convincing cause for the development of the hallmark symptoms of preeclampsia. The hypothesis of an agonistic autoimmune response to the AT1 receptor is presented. Immunoglobulin fractions from normotensive and preeclampsia patients were prepared for experimental tests. Model systems were tested in three categories to determine if AT 1 receptor specific activation and receptor-ligand interaction was caused by a suspected autoantibody. Activation was found in rat neonatal cardiornyocytes that caused an increased contraction rate. This activity was found in preeclampsia patients, absent in normotensive patients. The activation was antagonized by losartan, an AT1 receptor antagonist, and by epitope peptide competition of the receptor-ligand type interaction. This epitope was the 7 amino acid peptide fragment, AFHYESQ, a sequence present in the second extracellular loop of the AT1 receptor. The patterns of AT1 receptor activation were also found in a human trophoblast cell line, HTR8, with an effect on Pai-1 secretion, a factor that plays a role in preventing hypercoagulation. In human mesangial cells, the AT1 receptor autoantibody present in the immunoglobulin fraction from preeclampsia patients was found to stimulate the secretion of Pai-1, and IL-6, a factor that plays a role in the activation of an inflammatory response. This activity was found in samples from preeclampsia patients, but absent in normotensive patients. Tests including losartan, AFHYESQ, and a non-competitive peptide demonstrated that the secretion of Pai-1 and IL-6 met the criteria for AT1 receptor activation by the suspected agonistic autoantibody. These three model systems address relevant pathophysiology for preeclampsia patients, including increased cardiac output, abnormal placentation, and renal damage. The AT1 receptor agonistic autoantibody is potentially a key player in the development of the pathology and symptoms of preeclampsia. ^

Arachidonic acid mediates angiotensin II effects on p21ras in renal proximal tubular cells via the tyrosine kinase-Shc-Grb2-Sos pathway

In kidney epithelial cells, an angiotensin II (Ang II) type 2 receptor subtype (AT2) is linked to a membrane-associated phospholipase A2 (PLA2) and the mitogen-activated protein kinase (MAPK) superfamily. However, the intervening steps in this linkage have not been determined. The aim of this study was to determine whether arachidonic acid mediates Ang II’s effect on p21ras and if so, to ascertain the signaling mechanism(s). We observed that Ang II activated p21ras and that mepacrine, a phospholipase A2 inhibitor, blocked this effect. This activation was also inhibited by PD123319, an AT2 receptor antagonist but not by losartan, an AT1 receptor antagonist. Furthermore, Ang II caused rapid tyrosine phosphorylation of Shc and its association with Grb2. Arachidonic acid and linoleic acid mimicked Ang II-induced tyrosine phosphorylation of Shc and activation of p21ras. Moreover, Ang II and arachidonic acid induced an association between p21ras and Shc. We demonstrate that arachidonic acid mediates linkage of a G protein-coupled receptor to p21ras via Shc tyrosine phosphorylation and association with Grb2/Sos. These observations have important implications for other G protein-coupled receptors linked to a variety of phospholipases.

Aminopeptidase A inhibitors as potential central antihypertensive agents

Overactivity of the brain renin-angiotensin system (RAS) has been implicated in the development and maintenance of hypertension in several experimental models, such as spontaneously hypertensive rats and transgenic mice expressing both human renin and human angiotensinogen transgenes. We recently reported that, in the murine brain, angiotensin II (AngII) is converted to angiotensin III (AngIII) by aminopeptidase A (APA), whereas AngIII is inactivated by aminopeptidase N (APN). If injected into cerebral ventricles (ICV), AngII and AngIII cause similar pressor responses. Because AngII is metabolized in vivo into AngIII, the exact nature of the active peptide is not precisely determined. Here we report that, in rats, ICV injection of the selective APA inhibitor EC33 [(S)-3-amino-4-mercaptobutyl sulfonic acid] blocked the pressor response of exogenous AngII, suggesting that the conversion of AngII to AngIII is required to increase blood pressure (BP). Furthermore, ICV injection, but not i.v. injection, of EC33 alone caused a dose-dependent decrease in BP by blocking the formation of brain but not systemic AngIII. This is corroborated by the fact that the selective APN inhibitor, PC18 (2-amino-4-methylsulfonyl butane thiol), administered alone via the ICV route, increases BP. This pressor response was blocked by prior treatment with the angiotensin type 1 (AT1) receptor antagonist, losartan, showing that blocking the action of APN on AngIII metabolism leads to an increase in endogenous AngIII levels, resulting in BP increase, through interaction with AT1 receptors. These data demonstrate that AngIII is a major effector peptide of the brain RAS, exerting tonic stimulatory control over BP. Thus, APA, the enzyme responsible for the formation of brain AngIII, represents a potential central therapeutic target that justifies the development of APA inhibitors as central antihypertensive agents.

Genetic transfer of a nonpeptide antagonist binding site to a previously unresponsive angiotensin receptor.

Mutational analysis based on the pharmacological differences between mammalian and amphibian angiotensin II receptors (AT receptors) previously identified 7 aa residues located in transmembrane domains (TMs) III (Val-108), IV (Ala-163), V (Pro-192, Thr-198), VI (Ser-252), and VII (Leu-300, Phe-301) of the rat AT receptor type 1b (rAT1b receptor) that significantly influenced binding of the nonpeptide antagonist Losartan. Further studies have shown that an additional 6 residues in the rAT1b receptor TMs II (Ala-73), III (Ser-109, Ala-114, Ser-115), VI (Phe-248), and VII (Asn-295) are important in Losartan binding. The 13 residues required for Losartan binding in the mammalian receptor were exchanged for the corresponding amino acids in the Xenopus AT receptor type a (xATa receptor) to generate a mutant amphibian receptor that bound Losartan with the same affinity as the rAT1b receptor (Losartan IC50 values: rAT1b, 2.2 +/- 0.2 nM: xATa, > 50 microM; mutant, 2.0 +/- 0.1 nM). To our knowledge, this is the first report of a gain-of-function mutant in which the residues crucial to formation of a ligand binding site in a mammalian peptide hormone receptor were transferred to a previously unresponsive receptor by site-directed mutagenesis. Ala substitutions and comparison of mammalian and amphibian combinatorial mutants indicated that TM III in the rAT1b receptor plays a key role in Losartan binding. Identification of residues involved in nonpeptide ligand binding will facilitate studies aimed at elucidating the chemical basis for ligand recognition in the AT receptor and peptide hormone receptors in general.

Tonic stimulation of renin gene expression by nitric oxide is counteracted by tonic inhibition through angiotensin II.

This study was designed to examine the possible involvement of prostaglandins and nitric oxide (NO) in the renin stimulatory effect of angiotensin II (AngII) antagonists. To this end, plasma renin activities (PRAs) and renal renin mRNA levels were assayed in rats that were treated with the Ang-converting enzyme inhibitor ramipril or with the AngII AT1-receptor antagonist losartan. Ramipril and losartan increased PRA values from 7.5 +/- 1.6 to 86 +/- 6 and 78 +/- 22 ng of AngI per h per ml and renin mRNA levels from 112 +/- 9% to 391 +/- 20% and 317 +/- 10%, respectively. Inhibition of prostaglandin formation with indomethacin did not influence basal or ramipril-affected PRA. Basal renin mRNA levels also were unchanged by indomethacin, while increases in renin mRNA levels after ramipril treatment were slightly reduced by indomethacin. Inhibition of NO synthase by nitro-L-arginine methyl ester (L-NAME) reduced PRA values to 3.2 +/- 0.9, 34 +/- 13, and 12.1 +/- 2.7 ng of AngI per h per ml in control, ramipril-treated, and losartan-treated animals, respectively. Renin mRNA levels were reduced to 77 +/- 14% under basal conditions and ramipril- and losartan-induced increases in renin mRNA levels were completely blunted after addition of L-NAME. The AngII antagonists, furthermore, induced an upstream recruitment of renin-expressing cells in the renal afferent arterioles, which was also blunted by L-NAME. These findings suggest that renin mRNA levels are tonically increased by NO and that the action of NO is counteracted by AngII.

Mécanismes moléculaires de l’hypertrophie vasculaire dans le modèle animal d’hypertension essentielle (SHR)

La voie de signalisation des phosphoinositides joue un rôle clé dans la régulation du tonus vasculaire. Plusieurs études rapportent une production endogène de l’angiotensin II (Ang II) et de l’endothéline-1 (ET-1) par les cellules musculaires lisses vasculaires (CMLVs) de rats spontanément hypertendus (spontaneously hypertensive rats : SHR). De plus, l’Ang II exogène induit son effet prohypertrophique sur les CMLVs selon un mécanisme dépendant de la protéine Gqα et de la PKCẟ. Cependant, le rôle de l’axe Gqα/PLCβ/PKCẟ dans l’hypertrophie des CMLVs provenant d’un modèle animal de l’hypertension artérielle n’est pas encore étudié. L’objectif principal de cette thèse est d’examiner le rôle de l’axe Gqα/PLCβ1 dans les mécanismes moléculaires de l’hypertrophie des CMLVs provenant d’un modèle animal d’hypertension artérielle essentielle (spontaneously hypertensive rats : SHR). Nos premiers résultats indiquent que contrairement aux CMLVs de SHR âgés de 12 semaines (absence d’hypertrophie cardiaque), les CMLVs de SHR âgés de 16 semaines (présence d’hypertrophie cardiaque) présentent une surexpression protéique endogène de Gqα et de PLCβ1 par rapport aux CMLVs de rats WKY appariés pour l’âge. L’inhibition du taux d’expression protéique de Gqα et de PLCβ1 par des siRNAs spécifiques diminue significativement le taux de synthèse protéique élevé dans les CMLVs de SHR. De plus, la surexpression endogène des Gqα et PLCβ1, l’hyperphosphorylation de la molécule ERK1/2 et le taux de synthèse protéique élevé dans les CMLVs de SHR de 16 semaines ont été atténués significativement par des antagonistes des récepteurs AT1 (losartan) et ETA (BQ123), mais pas par l’antagoniste du récepteur ETB (BQ788). L’inhibition pharmacologique des MAPKs par PD98059 diminue significativement la surexpression endogène de Gqα/PLCβ1 et le taux de synthèse protéique élevé dans les CMLVs de SHR. D’un côté, l’inhibition du stress oxydatif (par DPI, inhibiteur de la NAD(P)H oxidase, et NAC , molécule anti-oxydante), de la molécule c-Src (PP2) et des récepteurs de facteurs de croissance (AG1024 (inhibiteur de l’IGF1-R), AG1478 (inhibiteur de l’EGFR) et AG1295 (inhibiteur du PDGFR)) a permis d’atténuer significativement la surexpression endogène élevée de Gqα/PLCβ1 et l’hypertrophie des CMLVs de SHR. D’un autre côté, DPI, NAC et PP2 atténuent significativement l’hyperphosphorylation de la molécule c-Src, des RTKs (récepteurs à activité tyrosine kinase) et de la molécule ERK1/2. Dans une autre étude, nous avons aussi démontré que la PKCẟ montre une hyperphosphorylation en Tyr311 dans les CMLVs de SHR comparées aux CMLVs de WKY. La rottlerin, utilisée comme inhibiteur spécifique de la PKCẟ, inhibe significativement cette hyperphosphorylation en Tyr311 dépendamment de la concentration. L’inhibition de l’activité de la PKCẟ par la rottlerin a été aussi associée à une atténuation significative de la surexpression protéique endogène de Gqα/PLCβ1 et l’hypertrophie des CMLVs de SHR. De plus, l’inhibition pharmacologique de l’activité de la PKCẟ, en amont du stress oxydatif, a permis d’inhiber significativement l’activité de la NADPH, le taux de production élevée de l’ion superoxyde ainsi que l’hyperphosphorylation de la molécule ERK1/2, de la molécule c-Src et des RTKs. À notre surprise, nous avons aussi remarqué une surexpression protéique de l’EGFR et de l’IGF-1R dans les CMLVs de SHR à l’âge de 16 semaines. L’inhibition pharmacologique de l’activité de la PKCẟ, de la molécule c-Src et du stress oxydatif a permis d’inhiber significativement la surexpression protéique endogène de ces RTKs. De plus, l’inhibition de l’expression protéique de l’EGFR et de la molécule c-Src par des siRNA spécifiques atténue significativement le taux d’expression protéique élevé de Gqα et de PLCβ1 ainsi que le taux de synthèse protéique élevé dans les CMLVs de SHR. Des siRNAs spécifiques à la PKCẟ ont permis d’atténuer significativement le taux de synthèse protéique élevé dans les CMLVs de SHR et confirment le rôle important de la PKCẟ dans les mécanismes moléculaires de l’hypertrophie des CMLVs selon une voie dépendante du stress oxydatif. En conclusion, ces résultats suggèrent un rôle important de l’activation endogène de l’axe Gqα-PLCβ-PKCẟ dans le processus d’hypertrophie vasculaire selon un mécanisme impliquant une activation endogène des récepteurs AT1/ETa, de la molécule c-Src, du stress oxidatif, des RTKs et des MAPKs.

Angiotensin II induces soluble fms-Like tyrosine kinase-1 release via calcineurin signaling pathway in pregnancy

Maternal endothelial dysfunction in preeclampsia is associated with increased soluble fms-like tyrosine kinase-1 (sFlt-1), a circulating antagonist of vascular endothelial growth factor and placental growth factor. Angiotensin II (Ang II) is a potent vasoconstrictor that increases concomitant with sFlt-1 during pregnancy. Therefore, we speculated that Ang II may promote the expression of sFlt-1 in pregnancy. Here we report that infusion of Ang II significantly increases circulating levels of sFlt-1 in pregnant mice, thereby demonstrating that Ang II is a regulator of sFlt-1 secretion in vivo. Furthermore, Ang II stimulated sFlt-1 production in a dose- and time-dependent manner from human villous explants and cultured trophoblasts but not from endothelial cells, suggesting that trophoblasts are the primary source of sFlt-1 during pregnancy. As expected, Ang II-induced sFlt-1 secretion resulted in the inhibition of endothelial cell migration and in vitro tube formation. In vitro and in vivo studies with losartan, small interfering RNA specific for calcineurin and FK506 demonstrated that Ang II-mediated sFlt-1 release was via Ang II type 1 receptor activation and calcineurin signaling, respectively. These findings reveal a previously unrecognized regulatory role for Ang II on sFlt-1 expression in murine and human pregnancy and suggest that elevated sFlt-1 levels in preeclampsia may be caused by a dysregulation of the local renin/angiotensin system.

Étude de la modulation de l'activité du système nerveux sympathique dans les maladies cardio-vasculaires : rôle du système rénine angiotensine et du monoxyde d'azote

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Étude de la modulation de l'activité du système nerveux sympathique dans les maladies cardio-vasculaires : rôle du système rénine angiotensine et du monoxyde d'azote

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Angiotensin receptor I stimulates osteoprogenitor proliferation through TGFβ-mediated signaling

Clinical studies of large human populations and pharmacological interventions in rodent models have recently suggested that anti-hypertensive drugs that target angiotensin II (Ang II) activity may also improve loss of bone mineral density. Here we identified in a genetic screen the Ang II type I receptor (AT1R) as a potential determinant of osteogenic differentiation and, implicitly, bone formation. Silencing of AT1R expression by RNA interference severely impaired the maturation of a multipotent mesenchymal cell line (W20-17) along the osteoblastic lineage. The same effect was also observed after the addition of the AT1R antagonist losartan but not the AT2R inhibitor PD123,319. Additional cell culture assays traced the time of greatest losartan action to the early stages of W20-17 differentiation, namely during cell proliferation. Indeed, addition of Ang II increased proliferation of differentiating W20-17 and primary mesenchymal stem cells and this stimulation was reversed by losartan treatment. Cells treated with losartan also displayed an appreciable decrease of activated (phosphorylated)-Smad2/3 proteins. Moreover, Ang II treatment elevated endogenous transforming growth factor β (TGFβ) expression considerably and in an AT1R-dependent manner. Finally, exogenous TGFβ was able to restore high proliferative activity to W20-17 cells that were treated with both Ang II and losartan. Collectively, these results suggest a novel mechanism of Ang II action in bone metabolism that is mediated by TGFβ and targets proliferation of osteoblast progenitors.

Internal mammary artery smooth muscle cells resist migration and possess high antioxidant capacity

Objective- This study investigated whether differences exist in atherogen-induced migratory behaviors and basal antioxidant enzyme capacity of vascular smooth muscle cells (VSMC) from human coronary (CA) and internal mammary (IMA) arteries. Methods- Migration experiments were performed using the Dunn chemotaxis chamber. The prooxidant [NAD(P)H oxidase] and antioxidant [NOS, superoxide dismutase, catalase and glutathione peroxidase] enzyme activities were determined by specific assays. Results- Chemotaxis experiments revealed that while both sets of VSMC migrated towards platelet-derived growth factor-BB (1-50 ng/ml) and angiotensin II (1-50 nM), neither oxidized-LDL (ox-LDL, 25-100 �g/ml) nor native LDL (100 �g/ml) affected chemotaxis in IMA VSMC. However, high dose ox-LDL produced significant chemotaxis in CA VSMC that was inhibited by pravastatin (100 nM), mevastatin (10 nM), losartan (10 nM), enalapril (1 �M), and MnTBAP (a free radical scavenger, 50��M). Microinjection experiments with isoprenoids i.e. geranylgeranylpyrophosphate (GGPP) and farnesylpyrophosphate (FPP) showed distinct involvement of small GTPases in atherogen-induced VSMC migration. Significant increases in antioxidant enzyme activities and nitrite production along with marked decreases in NAD(P)H oxidase activity and O2 .- levels were determined in IMA versus CA VSMC. Conclusions- Enhanced intrinsic antioxidant capacity may confer on IMA VSMC resistance to migration against atherogenic agents. Drugs that regulate ox-LDL or angiotensin II levels also exert antimigratory effects.

Differential mechanisms of angiotensin II and PDGF-BB on migration and proliferation of coronary artery smooth muscle cells

Angiotensin II (Ang II) and platelet-derived growth factor-BB (PDGF-BB) are associated with excessive cell migration, proliferation and many growth-related diseases. However, whether these agents utilise similar mechanisms to trigger vascular pathologies remains to be explored. The effects of Ang II and PDGF-BB on coronary artery smooth muscle cell (CASMC) migration and proliferation were investigated via Dunn chemotaxis assay and the measurement of [3H]thymidine incorporation rates, respectively. Both atherogens produced similar degrees of cell migration which were dramatically inhibited by mevastatin (10 nM). However, the inhibitory effects of losartan (10 nM) and MnTBAP (a free radical scavenger; 50 μM) were found to be unique to Ang II-mediated chemotaxis. In contrast, MnTBAP, apocynin (an antioxidant and phagocytic NADPH oxidase inhibitor; 500 μM), mevastatin and pravastatin (100 nM) equally suppressed both Ang II and PDGF-BB-induced cellular growth. Although atherogens produced similar changes in NADPH oxidase, NOS and superoxide dismutase activities, they differentially regulated antioxidant glutathione peroxidase activity which was diminished by Ang II and unaffected by PDGF-BB. Studies with signal transduction pathway inhibitors revealed the involvement of multiple pathways i.e. protein kinase C, tyrosine kinase and MAPK in Ang II- and/or PDGF-BB-induced aforementioned enzyme activity changes. In conclusion, Ang II and PDGF-BB may induce coronary atherosclerotic disease formation by stimulating CASMC migration and proliferation through agent-specific regulation of oxidative status and utilisation of different signal transduction pathways.

Investigating Angiotensin II in cardiac remodelling and the counter-regulatory actions of Angiotensin-(1-9)

Cardiovascular diseases (CVDs) including, hypertension, coronary heart disease and heart failure are the leading cause of death worldwide. Hypertension, a chronic increase in blood pressure above 140/90 mmHg, is the single main contributor to deaths due to heart disease and stroke. In the heart, hypertension results in adaptive cardiac remodelling, including LV hypertrophy to normalize wall stress and maintain cardiac contractile function. However, chronic increases in BP results in the development of hypertensive heart disease (HHD). HHD describes the maladaptive changes during cardiac remodelling which result in reduced systolic and diastolic function and eventually heart failure. This includes ventricular dilation due to eccentric hypertrophy, cardiac fibrosis which stiffens the ventricular wall and microvascular rarefaction resulting in a decrease in coronary blood flow albeit an increase in energy demand. Chronic activation of the renin-angiotensin-system (RAS) with its effector peptide angiotensin (Ang)II plays a key role in the development of hypertension and the maladaptive changes in HHD. Ang II acts via the angiotensin type 1 receptor (AT1R) to mediate most of its pathological actions during HHD, including stimulation of cardiomyocyte hypertrophy, activation of cardiac fibroblasts and increased collagen deposition. The counter-regulatory axis of the RAS which is centred on the ACE2/Ang-(1-7)/Mas axis has been demonstrated to counteract the pathological actions of Ang II in the heart and vasculature. Ang-(1-7) via the Mas receptor prevents Ang II-induced cardiac hypertrophy and fibrosis and improves cardiac contractile function in animal models of HHD. In contrast, less is known about Ang-(1-9) although evidence has demonstrated that Ang-(1-9) also antagonises Ang II and is anti-hypertrophic and anti-fibrotic in animal models of acute cardiac remodelling. However, so far it is not well documented whether Ang-(1-9) can reverse established cardiac dysfunction and remodelling and whether it is beneficial when administered chronically. Therefore, the main aim of this thesis was to assess the effects of chronic Ang-(1-9) administration on cardiac structure and function in a model of Ang II-induced cardiac remodelling. Furthermore, this thesis aimed to investigate novel pathways contributing to the pathological remodelling in response to Ang II. First, a mouse model of chronic Ang II infusion was established and characterised by comparing the structural and functional effects of the infusion of a low and high dose of Ang II after 6 weeks. Echocardiographic measurements demonstrated that low dose Ang II infusion resulted in a gradual decline in cardiac function while a high dose of Ang II induced acute cardiac contractile dysfunction. Both doses equally induced the development of cardiac hypertrophy and cardiac fibrosis characterised by an increase in the deposition of collagen I and collagen III. Moreover, increases in gene expression of fibrotic and hypertrophic markers could be detected following high dose Ang II infusion over 6 weeks. Following this characterisation, the high dose infusion model was used to assess the effects of Ang-(1-9) on cardiac structural and functional remodelling in established disease. Initially, it was evaluated whether Ang-(1-9) can reverse Ang II-induced cardiac disease by administering Ang-(1-9) for 2-4 weeks following an initial 2 week infusion of a high dose of Ang II to induce cardiac contractile dysfunction. The infusion of Ang-(1-9) for 2 weeks was associated with a significant improvement of LV fractional shortening compared to Ang II infusion. However, after 4 weeks fractional shortening declined to Ang II levels. Despite the transient improvement in cardiac contractile function, Ang-(1-9) did not modulate blood pressure, LV hypertrophy or cardiac fibrosis. To further investigate the direct cardiac effects of Ang-(1-9), cardiac contractile performance in response to Ang-(1-9) was evaluated in the isolated Langendorff-perfused rat heart. Perfusion of Ang-(1-9) in the paced and spontaneously beating rat heart mediated a positive inotropic effect characterised by an increase in LV developed pressure, cardiac contractility and relaxation. This was in contrast to Ang II and Ang-(1-7). Furthermore, the positive inotropic effect to Ang-(1-9) was blocked by the AT1R antagonist losartan and the protein kinase A inhibitor H89. Next, endothelial-to-mesenchymal transition (EndMT) as a novel pathway that may contribute to Ang II-induced cardiac remodelling was assessed in Ang II-infused mice in vivo and in human coronary artery endothelial cells (HCAEC) in vitro. Infusion of Ang II to mice for 2-6 weeks resulted in a significant decrease in myocardial capillary density and this was associated with the occurrence of dual labelling of endothelial cells for endothelial and mesenchymal markers. In vitro stimulation of HCAEC with TGFβ and Ang II revealed that Ang II exacerbated TGF-induced gene expression of mesenchymal markers. This was not correlated with any changes in SMAD2 or ERK1/2 phosphorylation with co-stimulation of TGFβ and Ang II. However, superoxide production was significantly increased in HCAEC stimulated with Ang II but not TGFβ. Finally, the role of Ang II in microvesicle (MV)-mediated cardiomyocyte hypertrophy was investigated. MVs purified from neonatal rat cardiac fibroblasts were found to contain detectable Ang II and this was increased by stimulation of fibroblasts with Ang II. Treatment of cardiomyocytes with MVs derived from Ang II-stimulated fibroblasts induced cardiomyocyte hypertrophy which could be blocked by the AT1R antagonist losartan and an inhibitor of MV synthesis and release brefeldin A. Furthermore, Ang II was found to be present in MVs isolated from serum and plasma of Ang II-infused mice and SHRSP and WKY rats. Overall, the findings of this thesis demonstrate for the first time that the actions of Ang-(1-9) in cardiac pathology are dependent on its time of administration and that Ang-(1-9) can reverse Ang II-induced cardiac contractile dysfunction by acting as a positive inotrope. Furthermore, this thesis demonstrates evidence for an involvement of EndMT and MV signalling as novel pathways contributing to Ang II-induced cardiac fibrosis and hypertrophy, respectively. These findings provide incentive to further investigate the therapeutic potential of Ang-(1-9) in the treatment of cardiac contractile dysfunction in heart disease, establish the importance of novel pathways in Ang II-mediated cardiac remodelling and evaluate the significance of the presence of Ang II in plasma-derived MVs.