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Overactivity of the renin-angiotensin system (RAS), both systemically and within organs, is associated with ageing and cardiovascular diseases. Localised brain RAS is dysregulated in Alzheimer's disease (AD). In this thesis, I investigated brain RAS signalling in normal ageing and AD in transgenic mouse models and human post-mortem brain tissue. In mice, ACE-1, ACE-2, (ELISA) and enzyme activity (FRET-based activity assay), and Ang-II and Ang-(1-7) levels (ELISA), were measured at different ages corresponding to the onset of pathology and cognitive decline. In humans, RAS gene (RT-PCR), protein, and enzyme activities were measured in the frontal and temporal cortex in a normal ageing cohort (n=101, 19-80y) and an AD cohort (n=60), stratified according to Braak tangle stage (BS), as a proxy of disease stage. In mice, ACE-2 declined with normal ageing across all four APP-transgenic models. Surprisingly, RAS markers were similar between transgenic and wild-type mice and were not exacerbated in-line with the onset of disease or cognitive decline (except for elevated ACE-2 activity and Ang-II levels at a younger age in the aggressive 5xFAD model). In humans, an age- and disease-related dysregulation of RAS signalling was observed. The age-related imbalance was driven by cRAS overactivation and differed from RAS dysregulation in AD. Notably in AD, ACE-1 enzyme activity was induced in the early stages of disease (BS III-IV) and MAS1 and IRAP's expressions (mediators of regulatory RAS signalling) were reduced. In conclusion, (i) age-related changes in brain RAS differ between mice and humans, (ii) age-related changes in human brain RAS are driven by cRAS signalling (iii) AD-related changes in RAS include early induction of ACE-1 activity and a reduction in the expression of rRAS genes in late-stage AD. Overall, RAS in normal ageing differs from dysregulated RAS in AD, offering a potential novel therapeutic target in the early stages of AD.

The renin angiotensin system (RAS) is highly conserved across vertebrates. However, until recently it has not been extensively explored in zebrafish, an important model organism suitable for developmental studies, high-resolution in vivo imaging, and genetic and chemical screens. In common with mammals, the adult zebrafish kidney contains specialised renin-expressing mural cells that contain granules indicative of the processing and regulated secretion of active renin. Due to the translucent nature of zebrafish larvae, the functional pronephros is easily accessible for real-time imaging and in vivo studies of the RAS. The primary aim of this project was to develop novel tools to help elucidate the function of renin and the role of the RAS in zebrafish. I designed a zebrafish renin-luciferase reporter transgene using a previously published promoter sequence to investigate the transcriptional regulation of renin in real-time. The expression vector was injected into fertilised WIK zebrafish eggs and successful integration of the plasmid into the zebrafish genome was demonstrated. An in vivo assay was designed to select for highest luciferase expressers, but bioluminescent imaging revealed that the high signal stemmed from the yolk sac of zebrafish. Yolk sac expression appeared to originate from episomal transcription of the injected plasmid and, in the F0 fish, masked potential bona fide expression from the renin-expressing cells of the pronephros. A new transgene was generated using two reporters, driven by the same promoter sequence and the two reporters were separated by a '2A' sequence which codes for a self-cleaving peptide. This zebrafish line was generated to permit selection of tg(ren:LUC-2A-mCherry) fish for the strongest mCherry signal originating from renin expressing cells. However, no expression was observed at the anterior mesenteric artery (AMA). Further investigation revealed that when generating novel transgenic zebrafish, the reporter proteins are transiently expressed in the yolk sac of F0 founder fish and the ectopic expression can influence studies relying on quantitative reporters. Current studies largely rely on the transcriptional activity of RAS components and are restricted to timepoint-specific observations. The lack of antibodies prevents the measurement of RAS proteins in zebrafish. The amino acid sequence for zebrafish angiotensinogen is known and by comparison to mammalian sequences, I identified the amino acid sequences for angiotensin (Ang) I and II. The production of zebrafish Ang I and II peptides by solid phase peptide synthesis provided standards for the development of assays for zebrafish angiotensins. I used these assays to demonstrate that Captopril, which in mammals prevents the conversion of Ang I to Ang II by inhibiting the Angiotensin Converting Enzyme (ACE), is active in zebrafish, and that its administration leads to a dramatic decrease in Ang II. In parallel I designed and synthesised a fluorescent resonant energy transfer (FRET) probe to enable zebrafish renin activity to be measured. Using various zebrafish models I was able to demonstrate dynamic changes in renin activity and measure these using the first renin zebrafish FRET probe. I characterised a renin knockout zebrafish, generated by CrispRCas9 gene editing. Knockouts were identified by DNA sequencing which identified an 8bp deletion in exon 2 of the renin gene, causing a frameshift mutation and early termination of renin translation. Renin knockout fish proved to be viable and were screened for a phenotype using the high throughput automated screening system (VAST) which permits the precise imaging of a large number of live fish. Imaging revealed delayed development of the swim bladder and reduced fish length compared to age-matched controls. This was indicative of an overall developmental delay. The knockout fish were intercrossed with existing renal reporter lines to investigate phenotypes at a cellular level. Ren−/− tg(wt1b:EGFP) fish showed a delay in glomerular fusion of the pronephric kidney and a cross of the ren−/− with tg(ren:RFP-LifeAct) fish indicated a dramatic increase in renin-expressing cell along the renal mesonephric vasculature. Moreover, morphological examination revealed vacuolation of proximal tubules in the mesonephric kidney. The intercrossing of the ren−/− zebrafish to the tg(ren:RFP-LifeAct) and tg(acta2:EGFP) fluorescent reporters lead to the optimisation of a FAC sorting protocol and resulted in the first zebrafish ren- and acta2-expressing cells to be cultured and imaged using high-resolution microscopy imaging. In summary, my work has led to the first successful measurement of zebrafish AngI and AngII in mesonephric zebrafish tissue to demonstrate the effectiveness of Captopril in zebrafish as well as the first development of a zebrafish renin FRET probe to allow the measurement and quantification of renin activity in zebrafish, enabling more accurate studies of the zebrafish RAS. Lastly, successful phenotypic characterisation of the ren−/− fish will further our understanding of the role of renin and the RAS in zebrafish.

The apelin-apelin receptor (APLNR) system is an important regulator of cardiovascular homeostasis both in health and disease. Principal actions of the apelin-APLNR system are positive inotropism, vasodilatation, diuresis and a potential anti-inflammatory role in vascular tissue. The significance of this system is highlighted in heart failure and pulmonary hypertension. Preclinical models of these diseases report downregulation of apelin- APLNR, whilst knockout strains develop more severe phenotypes, more rapidly. Moreover treatment with exogenous apelin retards or prevents disease progression. In man plasma apelin concentrations are reduced in heart failure and vary with disease severity. Initial increases are reported in mild heart failure suggesting a compensatory role, but are depressed in severe heart failure. Limited data profile myocardial APLNR expression in heart failure and in keeping with plasma apelin concentrations, expression is reduced in severe heart failure. Of interest, the APLNR most closely resembles the angiotensin II type 1 receptor (AT1R), sharing similar tissue expression and sequence homology, but mediates opposing physiological actions. Furthermore, emerging preclinical data support receptor interactions between the APLNR and AT1R that modify their native signalling pathways. It is likely that the apelin-APLNR system serves to antagonise the renin-angiotensin system. Given the established role of angiotensin II, arguably the most important peptide in cardiovascular pathophysiology, any system influencing its actions merits further investigation. Current clinical studies are limited to 20 minutes infusions and understanding its cardiovascular effects requires more prolonged administration. There are concerns of tachyphylaxis and interaction with the renin-angiotensin-aldosterone system (RAAS), possibly reducing efficacy of APLNR agonism in clinical settings. In a series of randomised, blinded crossover clinical trials 60 healthy volunteers and 20 patients with chronic stable heart failure were enrolled to assess the effects of (Pyr1)apelin-13 infusion at rest, during acute and subacute infusion, exercise and upregulation of the renin-angiotensin system. I have identified that APLNR agonism is unaffected by prevailing levels of angiotensin II activity in local vascular beds and systemic haemodynamic infusions. Furthermore, the efficacy of (Pyr1)apelin-13 is retained in healthy volunteers and patients with chronic stable heart failure during acute and subacute infusions. Finally, systemic (Pyr1)apelin-13 does not alter exercise performance in healthy individuals. My findings support a role in targeting the APLNR in chronic heart failure and predict that efficacy will be retained in chronic dosing. Future research directed at other patient groups with ventricular dysfunction is merited, in order to further characterise the utility of this system. These studies are encouraging; however, longer term studies may reveal effects beyond haemodynamic alterations and examine the effects on cardiac fibrosis and endothelial function. A long acting agonist is required to fully evaluate the role of APLNR signalling in cardiovascular disease.

The vascular endothelium is intimately related to both the structure and function of blood vessels. Within this thesis, the role of the vascular endothelium in endogenous fibrinolysis is studied. In particular, the relationship of this critical endothelial function to the renin-angiotensin and kallikrein-kinin systems is addressed, as well as the influence of homocysteine and antioxidants. Angiotensin-converting enzyme (ACE) inhibition is known to be beneficial in heart failure and other cardiovascular diseases, and one of the beneficial effects may be a reduction in thrombotic events. Both angiotensin II and bradykinin may play a role in the regulation of endogenous fibrinolysis and both are modified by ACE inhibition, with decreased production of angiotensin II but decreased breakdown of bradykinin. The technique of bilateral forearm plethysmography was utilised to make forearm blood flow measurements, whilst allowing for the venous sampling of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor type-1 (PAI-1), not only from an arm infused with vasoactive peptides, but also from the non-infused arm which acts as a contemporaneous control. Studies were performed in healthy volunteers. Firstly, the effects of angiotensin II and bradykinin were studied. Angiotensin II did not affect acute endothelial cell t-PA or PAI-1 release. In contrast, bradykinin is a potent vasodilator and selective stimulus for acute local t-PA release. We went on to study how these effects were modified in healthy volunteers taking quinapril 40 mg daily, losartan 50 mg daily or placebo for the 7 days prior to forearm blood flow studies. We demonstrated a selective and marked augmentation of bradykinin-induced t-PA release during ACE inhibition which was absent during placebo and losartan therapy. These findings suggest that the beneficial clinical and vascular effects of ACE inhibition may in part be mediated through local augmentation of bradykinin-induced t-PA release. Finally, elevated plasma homocysteine measurements are associated with clinical thrombotic events, including myocardial infarction, and it is thought that an oxidative stress may be associated with hyperhomocysteinaemia. We hypothesised that experimental hyperhomocysteinaemia, produced by methionine-loading, would impair substance P-induced forearm vasodilatation and t-PA release. We further hypothesised that vitamin C supplementation might reverse these potential derangements in endothelial function. However, we showed that in healthy volunteers, t-PA release was augmented during methionine-loading and unaffected by vitamin C supplementation. This suggests that the acute endogenous fibrinolytic capacity of the endothelium is augmented during acute hyperhomocysteinaemia in healthy humans via an oxidation-independent mechanism. Chronic hyperhomocysteinaemia, however, may have very different effects.

Non-alcoholic fatty liver disease (NAFLD) afflicts 25% of the world’s population and is projected to increase due to the increasing prevalence of diabetes and obesity. The metabolic syndrome (MetS) is a cluster of conditions that raise the risk of cardiovascular disease. A strong association exists between MetS and NAFLD, and these two conditions share many characteristics (e.g., increased waist circumference, triacylglycerol, glucose, and blood pressure), along with insulin resistance, that promote their pathogenesis. The classical signaling pathway of the renin-angiotensin system (RAS) has been related to the development and progression of metabolic disease. The objectives of these projects were to elucidate the mechanisms associated with RAS that promote hepatic steatosis in a metabolic syndrome model and to investigate the effects of a nutrient overload via a glucose challenge. The first chapter describes the potential mechanisms by which chronic blockade of the classical RAS pathway decreased hepatic lipid accumulation and increased insulin sensitivity. The second chapter describes that blockade of the classical RAS pathway increased angiotensin 1-7 (Ang 1-7), which acts on the non-classical pathway of RAS. Ang 1-7 signaling is suggested to counteract RAS pathogenic signaling. The increased Ang 1-7 was associated with decreased hepatic lipid levels, triacylglycerol synthesis, fatty acid oxidation, gluconeogenesis, and improved glucose tolerance. Finally, the third chapter examined part of the redox status, which is a component of the “hit” hypotheses describing the pathogenesis of NAFLD, through the expression of hepatic glutathione enzymes. Chronic blockade of the classical RAS pathway decreased the expression of enzymes regulating glutathione metabolism (synthesis, usage, and reduction). Collectively, these data demonstrated that the classical RAS pathway contributes to the dysregulation of metabolism in the liver, which promotes the pathogenesis of NAFLD and MetS. Additionally, overactivation of RAS via AT1-mediated signaling may worsen the hepatic redox status, affecting liver glutathione regulation, which has systemic impact in controlling increased oxidant production. Because the mechanisms behind the development of NAFLD are not fully elucidated, understanding the molecules that dysregulate the metabolic and oxidative homeostasis may provide specific targets for the treatment and prevention of metabolic diseases.

Abstract: Increased renal venous pressure is a recognized risk factor for kidney dysfunction. It is not rare in clinical settings with venous congestion, such as heart failure, and is associated with worsening kidney function and increased mortality. However, the underling mechanisms are not well understood. In addition, advanced kidney dysfunction might result in sodium and fluid retention, which exacerbates the congestive state. Sodium handling at high renal venous pressure has not been well addressed. In this thesis, we first review principal mechanisms governing renal hemodynamics and sodium handling. Then we investigate how these mechanisms would relate to increased renal venous pressure. Pathophysiologically, both intravascular and extravascular factors can lead to an increased renal venous pressure. Limited data could be found regarding a comprehensive understanding of how increased renal venous pressure contributes to kidney dysfunction in disease states. Thus, it is not well understood how increased renal venous pressure contributes to renal dysfunction. It is therefore that we embarked on a number of studies to define the role of increased renal venous pressure on renal function. Increased renal venous pressure forms a physical factor that directly decreases renal blood flow. However, neurohumoral factors are likely involved in mediating effects of increased renal venous pressure on renal blood flow, glomerular filtration rate and sodium handling. To separate the role of renal venous pressure from the role of a congestive state as a whole, we adapted a model of isolated increased renal venous pressure in the rat. The acute isolated increase in renal venous pressure results in decreases in RBF, renal vascular conductance, glomerular filtration rate and heart rate. Two very powerful modulators are the renin-angiotensin system and renal sympathetic nerve activity. The roles of these two mechanisms in the renal response to increased renal venous pressure are not clearly delineated. Moreover, it has been suggested that the intrinsic mechanism intended to maintain renal blood flow and glomerular filtration rate stable upon fluctuations in renal perfusion pressure, renal autoregulation, is compromised by increased renal venous pressure, but this has hardly been studied in this context. Our data indicate that other than renal sympathetic nerve activity, renin-angiotensin system and autoregulation, have primary roles in vasoconstrictive response to renal venous pressure elevation. Furthermore, the supression of renal sympathetic nerve activity and inappropriate activation of the renin-angiotensin system contribute to the impact on heart rate and mean arterial pressure. Increased renal venous pressure also modulates the sodium handling. The inappropriate activation of renin-angiotensin system is also responsible for the sodium retention in renal venous pressure elevation. Increased renal venous pressure also tend to incease the tubular pressure, which might affect the tubular sodium tansport.In conclusion, increased renal venous pressure impairs kidney function, which involves potential modulators such as the renal sympathetic nerve activity, the renin-angiotensin system and others. This report describes an initial exploration of this area, with emphasis on the renin-angiotensin system, renal nerves and renal autoregulation.

Obesity is a risk factor for the development of pancreatic dysfunction, insulin resistance. Obesity-associated factors such as adipocyte dysfunction, inflammation, leptin resistance, and inappropriately activation of the renin-angiotensin system (RAS) contribute to the development of these disorders. RAS inhibition improves glucose homeostasis by improving pancreatic dysfunction, insulin resistance. The objectives of this project were to evaluate the mechanisms by which angiotensin receptor type 1(AT1) improves pancreatic dysfunction and insulin resistance. In the second chapter, we demonstrated that AT1 blockade before the development of hypertension normalized glucose-stimulated insulin secretion by increasing the circulating levels of glucagon-like peptide-1 (GLP-1) and by increasing the protein expression of pancreatic GLP-1 receptor. In the third chapter, we demonstrated that chronic AT1 blockade started after the development of hypertension decreased the abundance of large adipocytes, improved the ability to suppress non-esterified free fatty acids levels after an acute glucose challenge, decreased fasting plasma glucose, and protected against the worsening of glucose intolerance. The fourth chapter, we showed that that chronic AT1 blockade decreased hyperglycemia by enhancing adipose-specific insulin signaling and by decreasing the expression of genes involved in hepatic gluconeogenesis. Additionally, this study demonstrated that chronic AT1 inhibition increased the expression of CD36 protein in retroperitoneal fat and blunted the protein expression of fatty acid synthase in response to an acute glucose challenge, suggesting that AT1 blockade reduces the obesity-associated hepatic triglyceride accumulation by decreasing hepatic lipogenesis and decreasing the sequestration of free fatty acids from circulation. Our findings expand our knowledge of how inappropriately activated RAS contributes to the development of pancreatic dysfunction and insulin resistance. By understanding the mechanisms by which AT1 blockade improves these disorders, we will be able to identify new molecular targets to be used to develop better therapeutic approaches aimed at decreasing the prevalence of type 2 diabetes.

Abstract: Patients suffering from inflammatory disorders are at higher risk of cardiovascular (CV) morbidity and mortality compared to general population. Nonsteroidal anti-inflammatory drugs (NSAIDs) are used to treat pain and inflammation associated with inflammatory disorders such as rheumatoid arthritis (RA). However, since the withdrawal of rofecoxib from the market, due to reports of life threatening CV incidents in its users, other NSAIDs are also suspected for such risks. Combined effects of inflammation and NSAIDs use on CV/renal risks are not well understood. However, many hypotheses have been suggested to explain the mechanisms behind these risks, yet none is conclusive and further pharmacological and pharmacokinetic explanations are needed to be explored. The renin angiotensin system (RAS) and cytochrome P450 (CYP) metabolites of arachidonic acid (ArA) are two major systems responsible for maintaining CV homeostasis in the body. Both systems contain distinct components in their pathways which physiologically oppose each other enabling a naturel balance. RAS component includes angiotensin converting enzymes (ACE, ACE2), physiologically active peptides (Ang-II, Ang-(1-7)) and angiotensin receptors (AT1R, AT2R Mas). The ACE/Ang-II/AT1R are group into what is called as cardiotoxic axis and ACE2/Ang-(1-7)/Mas are group into what is called as cardioprotective axis. Similarly, CYP pathway consists of CYP-enzymes and their arachidonic acid (ArA) metabolites (i.e., 20-hydroxyeicosatetraenoic acid; 20-HETE and epoxyeicosatrienoic acids; EETs, respectively). They are grouped into hydroxylase pathway (i.e., CYP-hydroxylases and 20-HETE) which is cardiotoxic in nature and epoxygenase pathway (i.e., CYP-epoxygenases and EETs) which is cardioprotective in nature. Constitutively, a balance exists between these components. However, under inflammatory conditions as well as in CV diseases this balance is altered indicating cardiotoxicity. We hypothesized that NSAIDs induced CV/renal risks involve their effects on RAS and ArA systems in the heart and kidney tissues. Also the extent of tissue exposure to NSAIDs may govern such risks. To test these hypotheses we first performed a systematic review of randomized control trials and observational studies. Looking for published evidence that NSAIDs differ in their extent of CV/renal risks. Based on these findings we choose four NSAIDs (rofecoxib, celecoxib, meloxicam, and flurbiprofen) to be dosed in Sprague Dawley rats, to investigate their extent of tissue accumulation and how it affects RAS and ArA pathways in adjuvant arthritis (AA) rat model of inflammation. Our results suggest that rofecoxib have highest while meloxicam and celecoxib have least potential of causing CV toxicity in population. Rofecoxib and flurbiprofen were also found to have higher tissue accumulation compared to meloxicam which minimally distributes in heart and kidney tissues. Celecoxib was an exception, which have high tissues accumulation yet possess less CV risks. Our results in AA rats showed that inflammation results in lower ACE2/Ang-(1-7)/Mas the cardioprotective axis, over ACE/Ang-II/ AT1R cardiotoxic axis. NSAIDs restored the constitutive balance, perhaps due to their anti-inflammatory properties. However, difference exists in terms of NSAIDS effects on ArA metabolism. Rofecoxib and flurbiprofen when dosed in inflamed rats, further increased 20-HETE/EETs cardiotoxic/cardioprotective metabolites concentration in the plasma and heart of AA rats. But meloxicam and celecoxib were devoid of these effects. These findings also correspond to higher tissue accumulation and higher CV risk reported for rofecoxib and flurbiprofen compared to meloxicam. We concluded that inflammation has detrimental effects on both RAS and ArA metabolism. NSAIDs effects on RAS are anti-inflammatory and beneficial in nature, however, some NSAIDs alter ArA metabolism resulting in higher concentration of cardiotoxic metabolites in the body. Moreover, NSAIDs with higher tissue distribution (e.g., rofecoxib, flurbiprofen) are more likely to interfere with ArA metabolism and may pose higher CV risks. While, NSAIDs which minimally distributes (e.g., meloxicam) into heart and kidney tissues poses significantly safer cadiorenal profiles. We also found that plasma and heart profiles of ArA metabolites of are very similar to each other, thus plasma ArA metabolites can serve as surrogate biomarkers of NSAIDs induced cardiotoxicity.

Pathological activation of the renin-angiotensin system (RAS) and inflammation are associated with hypertension and the development of metabolic syndrome (MetS). The progression of MetS is also associated with the development of chronic kidney disease (CKD) and nonalcoholic fatty liver disease (NAFLD). However, the contributions of the angiotensin type 1 receptor (AT1) receptor activation and tumor necrosis factor (TNF)-α-mediated inflammation have on the development of insulin resistance and how they impact hepatorenal health is not well defined. The Otsuka Long-Evans Tokushima Fatty (OLETF) rat is a well-characterized model of diet-induced obesity that progresses similarly to humans. Using this model and its lean strain counterpart, the Long Evans Tokushima Otsuka (LETO) the progression of the disease was observed in six groups of 16 week old rats with 6 weeks of treatment: 1) lean strain-control LETO; n=5, 2) obese OLETF; n=7, 3) OLETF + angiotensin receptor blocker (ARB; 10 mg olmesartan/kg; n=8), 4) OLETF + TNF-α inhibitor (ETAN; 1.25mg etanercept/kgd; n=6), 5) OLETF + angiotensin receptor blocker + TNF-α inhibitor (ARB + ETAN; 10 mg olmesartan/kg + 1.25mg etanercept/kgd; n=6), and 6) OLETF + calcium channel blocker (CCB; 5 mg amlodipine/kg; n=7).All treatments resulted in similar reductions in blood pressure; however the acute removal of etanercept resulted in an increase in blood pressure that was immediately reduced again once reintroduced. CCB treatment was not able to improve glucose tolerance or insulin resistance index, while ARB, ETAN, and ARB + ETAN were. Similarly, improvements in adiposity were observed with all treatment groups. Renal albumin excretion and glutathione peroxidase was recovered only with ETAN treatment. Renal 4-hydroxynonenal was reduced with ARB treatment. Hepatic non-esterified fatty acids were reduced with all treatments, however, there was a greater reduction found with ETAN and ARB + ETAN treatments. Despite relatively similar reductions in systolic blood pressure and adiposity, the calcium channel blocker did not improve glucose tolerance or calculated insulin resistance index, while the other treatments did, demonstrating that improper RAS activation and inflammation are larger factors contributing to the development of impaired glucose and lipid metabolism and regulation during metabolic syndrome, than hypertension. These results suggest that AT1 activation and TNF-α mediated inflammation are mechanistically important in the development of metabolic syndrome and that the associated increase in systolic blood pressure (SBP) may be a consequence of the condition. They also suggest that there are other factors contributing to impaired glucose tolerance and adiposity since none of the treatments completely ameliorated the derangement.

Abstract: Crystalline glucosamine (GlcN) sulfate salt formulation is approved as a prescription drug with anti-inflammatory effects for the management of osteoarthritis (OA) in the European Union, while it is considered as a nutraceutical in North America. The effectiveness of GlcN for ameliorating the signs and symptoms of OA in humans is still the subject of scientific debate. In animal studies using high doses, its effectiveness is undisputable; however results of clinical trials in humans are controversial. One of the objectives of this thesis was to address the possible causes of controversy around GlcN effectiveness in humans, and to establish a minimum effective dose (MED) needed to produce minimum anti-inflammatory effective concentration in the rat adjuvant arthritis animal model. Many explanations have been offered as the root of the controversy including superiority of a crystalline sulfate salt over the hydrochloride form, industry bias, insensitive assessment metrics, and poor methodology. Herein, we rule out a difference in bioequivalence between GlcN salts and suggest additional factors; i.e., inconsistency in the GlcN content of some products used in trials, under-dosing of patients, as well as variable pharmacokinetic indices as possible reasons for the lack of GlcN efficacy observed in some studies. Clinical trials using high doses of pharmaceutical grade GlcN, or formulations with greater bioavailability should yield positive results. Our results indicate that the MED is between 40 to 80 mg/kg/day that generates a maximum plasma concentration in the range of 1.37 ± 0.24 to 5.31± 6.84 mg/L, close to the rang of concentration reported for pharmaceutical grades of GlcN in humans. GlcN efficacy is dose and concentration dependent and if the data could be extrapolated to humans, using a higher than the commonly tested 1500 mg/kg /day dosage regimen may provide more clear treatment outcomes. Inflammation influences the body as whole and imbalances the homeostasis of different regulatory systems in particular. Renin-angiotensin system (RAS) and arachidonic acid (ArA) pathway are two main regulators of cardiovascular (CV) systems which could be affected by inflammation. Other objectives of this thesis were to investigate if experimentally induced adjuvant arthritis (AA), as an animal model for systemic inflammation, alters different components of the RAS such as angiotensin converting enzymes (ACE) expression, angiotensin (Ang) peptides systemic and local concentration, and their associated receptors expression level. We also investigated the effect of AA on ArA metabolites concentration in the plasma, heart and kidney tissues. We correlated the RAS components and ArA metabolites concentration in order to identify reliable biomarkers for prediction of CV complications in vulnerable individuals suffering from inflammatory conditions such as rheumatoid arthritis (RA). GlcN with anti-inflammatory effects is a good candidate for modulating the detrimental effect of inflammation on the RAS and ArA pathways. We studied the impact of GlcN on these two systems. GlcN was able to reestablish the disturbed balance of the RAS and ArA pathway by reinstating the ratio of cardioprotective (vasodilator, anti-proliferative and anti-hypertrophic) over cardiotoxic (vasoconstrictive, proliferative and pro-hypertrophic) components of these systems. GlcN by presenting modulatory effects on the RAS and ArA pathways could be an alternative over NSAIDs which their long term use imposes CV side effects in RA patients.

There are more than 450,000 patients receiving chronic dialysis in the US. Patients with end stage renal disease are statutorily eligible for Medicare, regardless of age. Due to high prevalence of poverty, many are dually enrolled in Medicare and Medicaid and are automatically enrolled in Part D (prescription drug insurance). This dissertation comprises 3 studies involving prescription drug claims in dialysis patients. In the first study, I constructed and validated risk scores based on Part D claims in incident and prevalent dialysis patients. Comorbidity indices derived from administrative data are typically based on diagnosed diseases, but the middling sensitivity of diagnosis codes limits the accuracy of these indices. In contrast, prescription drug claims are bona fide evidence of medication dispensation. I investigated aspects of newly developed scores and compared their performance in predicting risk with older scores based on diseases. In the second study, I assessed the efficacy and safety of pharmacologic inhibition of the renin-angiotensin system in dialysis patients with congestive heart failure. ACE inhibitors and ARBs are recommended for the treatment of heart failure with reduced ejection fraction. However, pivotal clinical trials all excluded patients on dialysis. I used propensity score matching to identify matched controls for treated patients that were newly dispensed an ACE inhibitor or ARB shortly after discharge from hospitalization for heart failure. I described the relative hazards of mortality and morbidity with treatment. In the third study, I assessed relative hazards of death and hospitalization associated with 4 ACE inhibitors and 2 ARBs in dialysis patients with congestive heart failure. The existence of class effects is generally presumed, but not necessarily supported by evidence regarding clinical outcomes. I showed that the most widely used agent (lisinopril) in this patient population is not necessarily associated with best outcomes.

In spite of significant progress in our knowledge of mechanisms that control blood pressure, our understanding of the pathogenesis of hypertension, its genetics, and population efforts to control blood pressure, hypertension remains the leading risk factor for mortality worldwide. It’s estimated that 1 out of every 3 adults has hypertension. Hypertension is a major risk factor for cardiovascular disease and stroke, and is considered a primary or contributing cause of death to more than 2.4 million US deaths each year. Although spontaneous hypertension has been the subject of substantial research, many critical questions remain unanswered. To investigate mechanisms underlying spontaneous hypertension, a unique rodent breeding approach was used to isolate nuclear and mitochondrial genes contributing to the disease. By diluting the nuclear genome of the Spontaneously Hypertensive Rat on a normotensive Brown Norway background while maintaining the SHR mitochondrial genome, I investigated both intrinsic and extrinsic mechanisms of the kidney and its relationship to hypertension. Chapter 2 documents the dominance of the hypertensive phenotype in our rodent colony, despite the dilution of the nuclear genome of the SHR. Chapter 3 presents data indicating that the renin-angiotensin system, particularly the location and abundance of the AT1 receptor may play an important role in the manifestation of spontaneous hypertension. Chapter 4 presents that rats in our rodent colony exhibited normal pressure-natriuresis and kidney function; however, hypertensive rats had a reduced ability to sense orally ingested sodium chloride, thus necessitating chronic elevations of arterial pressure in order to maintain sodium balance. This chronic pressure-natriuresis relationship shifts the renal function curve to the right, thus sustaining elevated blood pressure. Chapter 5 presents data that genes important for oxidative phosphorylation may play a critical role in the development of hypertension. Both nuclear and mitochondrial oxidative phosphorylation genes were downregulated in hypertensive rats compared with normotensive rats. Data presented in every chapter highlights the importance of the kidney in the pathogenesis of hypertension. Humoral, genetic and genomic mechanisms of the kidney appear to play a dominant role in the development and maintenance of the disease.

Renin-angiotensin system (RAS) activation and phenotypic modulation of vascular smooth muscle cells (VSMCs) are common characteristics associated with human diseases, such as pulmonary hypertension, atherosclerosis and stroke. While elevation of the RAS hormone product, angiotensin II (AngII) is a well-established risk in these diseases; the mechanism of activation of RAS and modulation of VSMC phenotype by AngII is vague, suggesting that novel global regulators may mediate the risk by AngII. MicroRNAs (miRNAs) represent one such class of potential global regulators. MiRNAs are small (~22 nt), endogenous, non-coding RNAs that act as post-transcriptional regulators of physiological processes. MiRNAs primarily function by binding to complementary target sites in the 3’-untranslated regions (3’-UTR) of mRNAs, causing translational repression and/or mRNA destabilization. To elucidate the global miRNA expression profile following chronic Angiotensin II Type 1 Receptor (AT1R) activation by AngII, we performed microarray analysis in 23 biological and technical replicates derived from humans, rats and mice. We pharmacologically distinguished the AT1R-regulated miRNA profiles by comparing technical replicates treated with the specific AT1R-blocker, losartan and biological replicates following chronic AT2R activation by AngII. Thirty-two miRNAs are AngII-regulated universally. Most other miRNAs are regulated in a treatment- or species-specific manner. A few miRNAs are unique to specific cell types. We have previously shown that a single miRNA can have multiple targets, potentially providing simultaneous regulation of the genes involved in a physiological pathway and accounting for a complex phenotype, such as human heart failure (JBC 284: 27487-27499). In the current study, we explored the extent to which AngII/AT1R-regulated miRNAs contribute to maintenance of RAS homeostasis and phenotypic modulation of VSMCs.A distinct AngII-regulated miRNA expression pattern emerged in the human and rat VSM cell lines in the profiling experiment, which was validated in our independent samples. Of the 17 miRNAs comprising the VSMC expression pattern, we selected miR-483 as a representative candidate for further study because of its location within the genome and its ability to potentially target multiple components of RAS. In addition, we show evidence that suggests muscle cell-specific expression of miR-483. We incorporated a miR-483 expression cassette into two distinct cell lines as a means to determine post-transcriptional inhibition of specific RAS component gene expression. The functionality of miR-483, in terms of modulating the cellular phenotype, was assessed in a wound-healing assay. Overall, miR-483 regulates both RAS and AngII-activated migration; specifically in VSMCs. Our results further suggest that following inhibition of several AT1R-activated kinases, the AngII-regulated MEK1 kinase signaling cascade most effectively mediates the steady state pool of miRNAs, which includes downregulation of mir-483. In this context, JAK2 inhibition is somewhat effective. This results in activation of RAS and switching of the VSMC phenotype associated with pathological states in vivo, such as hypertension. Further insight into these mechanisms will be valuable for a greater understanding of AngII biology. In the context of determining the full capacity of RAS as an intrinsic regulatory system, AngII-regulated miRNAs will likely have a strong influence on cardiovascular disease.

Human angiotensinogen gene locus is associated with hypertension. Human angiotensinogen gene (hAGT) has -6A/G polymorphism and -6A (rs5051) allele is associated with increased blood pressure. We have found that hAGT gene has three additional SNPs. Variants -1670A, -1562C, and -1561T almost always occur with -6A haplotype (Hap -6A); and variants -1670G, -1562G, and -1561G almost always occur with -6G haplotype (Hap -6G). We have shown that these polymorphisms affect the binding of glucocorticoid receptor to the promoter. Reporter construct containing 1.8 Kb of the hAGT gene promoter with Hap -6A has 4 fold increased glucocorticoid induced promoter activity as compared to Hap -6G. Therefore, we have generated transgenic (TG) mice containing either Hap -6A or Hap -6G of the hAGT gene to study the regulation of the hAGT gene in an in vivo situation. We have reported that transgenic mice with Hap -6A of hAGT gene has increased plasma angiotensinogen levels and significantly elevated blood pressure as compared to the Hap -6G. Since hAGT is not cleaved by mouse renin, we have generated double transgenic mice with human renin and hAGT gene of either Hap -6A or Hap -6G. We hypothesize that these three additional SNPs in the promoter of Hap -6A may be predisposing to hypertension in response to increased glucocorticoid levels.TG mice with Hap -6A and Hap -6G were treated with dexamethasone (DEX) (10µg/day in drinking water) for 72 hours. Our Q-RTPCR results show that mice with Hap -6A have significant increase in the mRNA expression of hAGT in the liver and the kidney on treatment with DEX whereas no significant changes were observed in Hap -6G. Q-RTPCR results were confirmed by western blots showing significant increase in the DEX induced expression of the hAGT protein in the liver and the kidney of the TG mice with Hap -6A. DEX treatment also increased the plasma hAGT protein and Ang-II levels in TG mice with Hap -6A whereas no significant changes were observed in TG mice with Hap -6G. The systolic blood pressure (SBP) of TG mice containing Hap -6A increased by 12 mmHg on treatment with DEX as compared to 5mmHg in the mice with Hap -6G. We subjected these TG mice to increased dosage of DEX at 50µg/day and observed a dose dependent modulation of SBP. DEX dependent increase in the SBP was reduced by losartan.

Currently, it is reported that ~65% and 34% of the U.S. population is overweight and obese, respectively. Obesity is a major risk factor for cardiovascular disease. Overweight and obese individuals are also at an increased risk of developing hypertension. Whole-body insulin sensitivity is reduced in obesity, resulting in insulin resistance and increased risk of type 2 diabetes. One possible mechanism contributing to insulin resistance in obesity hypertension is renin-angiotensin system (RAS) overactivation. The RAS exhibits vasocontricting and sodium-retaining properties, yet in vivo and in vitro animal experiments suggest impairment of whole-body insulin sensitivity with increased angiotensin II (Ang II) exposure. Furthermore, evidence from clinical studies indicates Ang II receptor blockers (ARBs) may reduce the incidence of new-onset diabetes compared to other antihypertensive agents in at-risk hypertensive patients. However, it is unclear if whole-body insulin sensitivity is improved with Ang II receptor blockade in humans. Thus, we tested the hypothesis that 8-week Ang II receptor blockade with olmesartan would improve whole-body insulin sensitivity in overweight and obese individuals with elevated blood pressure (BP). Olmesartan was selected for the present study because it is devoid of partial PPARγ agonist activity. To test our hypothesis, intravenous glucose tolerance tests were performed to measure insulin sensitivity before and after control and ARB treatment in a randomized crossover manner. Because skeletal muscle tissue accounts for ~75-90% of insulin-stimulated glucose uptake, a secondary exploratory aim was to examine skeletal muscle inflammatory and collagen response in relation to insulin sensitivity during ARB treatment. No baseline differences were observed between treatments (P>0.05). Both systolic (-11.7 mmHg; P=0.008) and diastolic (-12.1 mmHg; P=0.000) BP were reduced with ARB treatment. Insulin sensitivity was not different between treatments (P>0.05). No correlates of insulin sensitivity were identified. In addition, skeletal muscle inflammatory and collagen gene expression did not change from pre- to post-ARB treatment (P>0.05). Our findings suggest that short-term RAS blockade in overweight and obese adults with elevated BP does not improve whole-body insulin sensitivity, despite a significant BP reduction. Further studies are needed to clarify the role of individual RAS blockers on insulin sensitivity during RAS inhibition in obesity hypertension.

Adipose tissue is well-recognized as an endocrine organ which secretes a variety of bioactive molecules, including angiotensin II and its precursor angiotensinogen (Agt). There is mounting evidence linking the adipose renin-angiotensin system (RAS) and diet to obesity and obesity-related disorders. However, research addressing dietary regulation and function of adipose RAS is limited, and the specific mechanisms by which PUFAs modulate the endocrine function of adipose tissue remain largely unclear. There are several potential mechanisms that may mediate PUFA effects on Agt, including toll-like receptor signalling, prostaglandins or PPAR-gamma. Thus, we propose to investigate whether PUFAs differentially modulate Agt expression and secretion and to examine possible mechanisms by which PUFA alter Agt expression using the 3T3-L1 cell line. Differentiated 3T3-L1 adipocytes were treated with arachidonic acid (AA), eicosapentaenoic acid (EPA), AA + EPA, or vehicle (C) for 48 hours. Results showed a significant increase in intracellular Agt protein following treatment with PUFAs. Agt secretion, however, was only increased by AA. Interestingly, there is a dose-dependent decrease in Agt protein levels by EPA suggesting that a minimum concentration of n-3 PUFAs is required to elicit an Agt response. Agt mRNA levels were measured by RT-PCR and results showed a significant increase in Agt mRNA in response to treatment with AA but not EPA. These findings suggest that Agt regulation by PUFAs is complex and occurs both post-transcriptionally and post-translationally. Changes in mRNA stability may account for the observed effects of PUFAs. Adipocytes were treated with the transcriptional inhibitor actinomycin D (Act D) and Agt mRNA expression was measured over time. Total RNA was also measured at each time point to ensure that Act D treatment was effectively decreasing transcription. Agt mRNA expression was not significantly altered by treatment with EPA while treatment with AA increased Agt mRNA levels. These results suggest that Agt mRNA stability is differentially increased by n-6 but not n-3 PUFAs. Although there are clear effects of AA on Agt secretion and mRNA stability, the signaling pathways mediating this response remain to be determined, and additional studies are necessary to further dissect the underlying mechanisms of this regulation.

2031-01-02

This thesis reports the effects of an altered intrauterine environment on the offspring s renal, cardiovascular and renin angiotensin systems. After a midgestational asphyxial episode in fetal sheep (30 min total umbilical cord occlusion at 90 days; term 150 days) the hydrops that resulted had not completely resolved by 130 days. While the heart and kidneys were apparently unaffected, the brain and lung weights were 37% and 50% lower than sham values, respectively and there were joint contractures. The effects of maternal renal disease on the offspring were investigated. Although in utero fetuses of subtotally nephrectomised ewes (STNx) had altered urine flow rates, sodium excretion, haematocrits, plasma chloride and plasma renin levels, by 1-2 weeks after birth these values in the lambs (STNxL) were similar to controls (ConL) under baseline conditions. Body weight and the weights of most organs were similar, including the kidney, in which glomerular number was normal. In the neonatal period, the lambs were subjected to four challenges: furosemide (2 mg/kg intravenous bolus), infusion of angiotensin II and phenylephrine, intravenous infusion of 0.15M saline (50 ml/kg over 30 min) and haemorrhage (20% estimated blood volume over 10 min). These challenges revealed evidence of programming of several aspects of the renal, cardiovascular and renin angiotensin systems in the STNx offspring. As young adults at 6 months of age, male and female offspring of STNx ewes were normotensive and had normal renal function. On a high salt diet (HSD, 0.17M NaCl in 8L water for 5-7days), female offspring of both groups did not become hypertensive. However, the STNx offspring must have retained salt and water as plasma sodium was increased and haematocrit was decreased. In the STNx offspring only, there was a relationship between glomerular filtration rate (GFR) and mean arterial pressure, indicating an inability to maintain a constant GFR in response to changes in arterial pressure.

Fibrous tissue accumulation is an integral feature of the adverse structural remodeling of cardiac tissue seen with hypertensive heart disease. Given the importance of fibrous tissue in leading to myocardial dysfunction and failure, noninvasive monitoring of myocardial fibrosis by use of serological markers of collagen turnover could prove a clinically useful tool, particularly given the potential for cardioprotective and cardioreparative pharmacological strategies. An emerging experimental and clinical experience holds promise for the use of radioimmunoassays of various serological markers of fibrillar collagen type I and type III turnover in arterial hypertension. More specifically, the measurement of serum concentrations of procollagen type I C-terminal propeptide (a peptide that is cleaved from procollagen type I during the synthesis of fibril-forming collagen type I) may provide indirect diagnostic information on both the extent of myocardial fibrosis and the ability of antihypertensive treatment to diminish collagen type I synthesis and reduce myocardial fibrosis. This approach represents an exciting and innovative strategy, and available data set the stage for larger trials, in which noninvasive measures of fibrosis in hypertensive heart disease could prove useful.