Objetivo: Avaliar o estado nutricional e o risco de doença cardiovascular (DCV) de mulheres no climatério. Metodologia: Estudo transversal, cuja população foi composta por 200 mulheres de 35 a 65 anos, agrupadas de acordo com a fase do climatério: pré-menopausa (PRÉ), perimenopausa (PERI) e pós-menopausa (PÓS). As mulheres do grupo PÓS foram divididas em dois grupos: sem terapia de reposição hormonal (S/TRH) e com TRH oral por no mínimo 12 meses (C/TRH). O estado nutricional foi avaliado segundo o índice de massa corporal (IMC) e o percentual de gordura corporal (%GC); o risco de DCV foi avaliado segundo a relação cintura/quadril (RCQ). O nível de significância utilizado nas análises foi alfa=5%. Resultados: A prevalência de pré-obesidade + obesidade, segundo o IMC, foi 65,4% (PRÉ), 70,9% (PERI) e 67,4% (PÓS), porém não houve diferença significativa entre os grupos. Comparado ao grupo C/TRH, houve maior proporção de mulheres obesas no grupo S/TRH (p=0,04), 21,4% e 48,4%, respectivamente. O %GC revelou sobrepeso e obesidade em 50,7% (PRÉ), 66,7% (PERI) e 57,4% (PÓS); 67,7% (S/TRH) e 54,8% (C/TRH) – essas diferenças não foram estatisticamente significativas. O risco de DCV foi considerado alto e muito alto para a maioria das mulheres: 90,7% (PRÉ), 95,8% (PERI) e 84,1% (PÓS); 90,3% (S/TRH) e 76,2% (C/TRH) e não houve diferença estatística entre os grupos. Conclusão: Grande parte das mulheres apresentou alto risco de afecções relacionadas à obesidade, como a DCV, justificando a atenção à mulher no climatério para a prevenção das principais causas de mortalidade nesse grupo. / Purpose: To evaluate the nutritional status and cardiovascular disease (CVD) risk of climacteric women. Methodology: Transversal study, composed by 200 women aged 35-65 years, grouped according to the climacteric stage: premenopause (PRE), perimenopause (PERI) and postmenopause (POS). The PÓS group was divided in two groups: with no hormonal replacement therapy (N/HRT) and with oral HRT for at least 12 months (W/HRT). The nutritional status was assessed by body mass index (BMI) and body fat percentage (BF%); the CVD risk was assessed by waist hip ratio (WHR). The significance level used for the analyses was alpha=5%. Results: According to BMI, the pre-obesity + obesity prevalences were 65,4% (PRÉ), 70,9% (PERI) e 67,4% (POS) and there was no significant difference among these groups. Compared with the group W/HRT, there was a higher rate (p=0,04) of obesity among women in the N/HRT group: 21,4% and 48,4%, respectively. The BF% showed overweight and obesity in 50,7% (PRE), 66,7% (PERI) and 57,4% (POS); 67,7% (N/TRH) and 54,8% (W/TRH), differences that were not statisticaly significant. In all groups there was a greater proportion of women considered with high and very high risk of CVD: 90,7% (PRE), 95,8% (PERI) and 84,1% (POS); 90,3% (N/TRH) and 76,2% (W/TRH) and the risk did not differ statisticaly among the groups. Conclusion: The majority of women had increased obesity-related and cardiovascular disease risk, emphasizing women´s health attention programms to the prevention of the leading mortality causes in this group.

Recently, it has emerged that the NO metabolites, nitrite and nitrate can be chemically reduced in vivo to biologically active nitric oxide (NO). This generation of NO is dependent on reduction of nitrate to nitrite by facultative anaerobes on the dorsal surface of the tongue, entry of the nitrite into the enterosalivary circuit, transit to the stomach, and absorption through the gut wall into the circulation. Conversion of nitrite to NO is then facilitated by vascular nitrite reductase enzymes. This nitrate-nitrite-NO pathway has been shown to exert a number of beneficial effects in healthy volunteers e.g. lowering of blood pressure, however whether this pathway is affected by cardiovascular disease (CVD) is currently unknown. Ozone chemiluminescence was used to determine and compare nitrite and nitrate levels in 2 models of CVD. To study atherosclerosis wild type (WT) and apolipoprotein E knock out (ApoE KO) mice were used and for hypertension wistar kyoto (WKY) rats as controls vs. spontaneously hypertensive rats (SHR). Assessment of nitrite reductase activity was conducted in the compartment which showed the most consistent differences in distribution, the red blood cell (RBC) and in homogenates of liver tissue. The impact of dietary nitrite and nitrate on distribution of the 2 anions throughout the cardiovascular system was assessed to determine the utility of this approach in restoring levels of these anions in CVD. Finally, using flow cytometry I investigated whether dietary nitrate supplementation could be used to influence inflammatory responses as a mechanism to improve CVD. Compared to WT mice, nitrate levels were reduced in ApoE KO mice in the plasma and across most of the tissues. In contrast in SHRs, reduction of the anions was only apparent in RBCs with no differences compared to WKY in all other tested tissues. Furthermore I have demonstrated that the most efficient way to restore nitrate levels back up to baseline is through a dietary nitrate strategy and that a dose of 15mM nitrate in the drinking water is sufficient to achieve this. In addition I have shown that nitrite reductase activity is enhanced in CVD particularly at the level of the RBC in both atherosclerosis and hypertension and that this enhanced activity is due, in part, to upregulation of xanthine oxidoreductase (XOR). Finally I have shown that dietary nitrate is an effective way to modulate an acute inflammatory response. This modulation is mediated through interfering with the ability of the neutrophil to firmly adhere to the vascular endothelium. These changes were shown to be dose-dependent and concomitant with dose-dependent increases in plasma nitrite and plasma nitrate. These data suggest that utilization of the nitrate-nitrite-NO pathway with dietary nitrate may represent an effective approach for the treatment of CVD.

The incidence and mortality from cardiovascular disease (CVD) in patients with chronic kidney disease (CKD) far exceeds that seen in the general population. Whilst a number of risk factors and associations have been identified in patients with CKD that may contribute to the increased risk of CVD, our understanding of the underlying pathophysiology remains poor. It has previously been reported that uraemic animals sustain larger myocardial infarcts and that this ‘reduced ischaemia tolerance’ may in part explain the excess mortality from CVD seen in CKD patients. The aim of this work was to establish an in vivo model of uraemic myocardial infarction in order to further explore the pathophysiology of uraemic CVD with particular focus on ameliorating myocardial ischaemia-reperfusion injury using ischaemic and pharmacological preconditioning. An increase in myocardial infarct size was demonstrated in the sub-total nephrectomy model of chronic uraemia, confirming previous reports in the literature. However, infarct size was not found to be increased in adenine diet induced renal failure. In addition, it was demonstrated for the first time, that the techniques of ischaemic preconditioning (IPC) and remote ischaemic preconditioning (RIPC) are both efficacious and not attenuated by chronic uraemia induced by sub-total nephrectomy or adenine diet (IPC only). Investigations were undertaken using an agent (a HIF stabiliser, FG4497) to induce pharmacological preconditioning in both animals with renal insufficiency and those without. These studies demonstrate that stabilisation of hypoxia inducible factor (HIF) may be a promising strategy to induce pharmacological preconditioning. It is hoped that this work may lay the foundations for future investigations to determine why sub-totally nephrectomised rats have larger infarcts whilst those with adenine induced renal failure, with a substantially greater degree of renal dysfunction, do not. Moreover, it is hoped that; by demonstrating that uraemia 3 does not prevent or attenuate the myocardial protection afforded by ischaemic preconditioning, the recruitment of patients with CKD will be encouraged to clinical trials of both ischaemic preconditioning and other therapies to limit myocardial infarction.

Cardiovascular disease is the leading cause of death in patients with underlying chronic kidney disease (CKD). Up to one third of patients presenting with an acute coronary syndrome have CKD stage 3-5. Outcomes following acute myocardial infarction in patients with underlying CKD remain poor. CKD patients are routinely excluded from clinical trials in novel cardioprotective strategies resulting in a paucity of prospective data on which to base guidelines for clinical practice. The aims of this work were to: • Establish and characterise two models of chronic uraemia in rodents: the subtotal nephrectomy model and the adenine diet model. • Determine the effects of underlying chronic uraemia on myocardial ischaemia tolerance. • Examine pharmacological cardioprotective strategies in the context of underlying uraemia using a PARP inhibitor • Investigate the cardioprotective effects of ischaemic conditioning in the context of uraemia. Ischaemic preconditioning and postconditioning protocols were used in both uraemic and non-uraemic animals in a model of acute myocardial infarction. • Preliminary work, using standard molecular biological techniques, was carried out in order to confirm the putative survival pathways responsible for the effect of preconditioning. • Investigate the effect of combining early and late remote ischaemic preconditioning to identify whether summation of these strategies could provide additional tissue protection in a model of acute kidney injury. The results demonstrate that both models develop a uraemic phenotype. Subtotal nephrectomy animals exhibit reduced ischaemia tolerance. PARP inhibition as a pharmacological post conditioning agent was shown to be ineffective at conferring tissue protection, whereas both ischaemic preconditioning and postconditioning were effective cytoprotective strategies in both non-uraemic and uraemic animals. Furthermore, additional benefit was seen when early and late remote preconditioning were summated in a rodent model of acute kidney injury. This work provides a basis for future clinical trials in cardioprotection in the context of underlying CKD.

Cardiovascular disease is the leading cause of unnatural death worldwide. Damage to the endothelial glycocalyx impairs endothelial functions and thereafter leads to the development of cardiovascular diseases. Despite this, many issues remain to be explored in our understanding of the metabolism and vasculoprotective potential of the glycocalyx. This study focuses on the recovery and structural stability of the glycocalyx, and its role in electric field-directed cell migration in vitro. The integrity of the glycocalyx is compromised following trypsin treatment during cell passages. Results from our study show that cell seeding density affects the recovery speed of the glycocalyx in the first 48h. Higher cell density results in more rapid recovery of the glycocalyx. Regardless of the initial cell seeding density, a well-developed glycocalyx layer is observed when cell confluence is reached. Micropatterning is used to study effects of the cell shape on the recovery of the glycocalyx. Elliptical patterns have been used to conform endothelial cells to torpedo shapes, mimicking their morphology under a shear flow. More rapid development of the glycocalyx on elliptical cells is observed than that on circular shaped cells during the early stage of recovery. Effects of the actin cytoskeleton on the stability of the glycocalyx are investigated, following our interest in shedding of the glycocalyx in abnormal vascular microenvironment. Rapid depolymerisation of the actin cytoskeleton leads to cell retraction within 10mins, with the glycocalyx preserved on the cell surface. This is also seen during 24h persistent actin disruption under static conditions. However, when endothelial cells are subjected to 24h steady laminar shear stress, the glycocalyx is seen to shift to the downstream of the cell surface in the control group, and with actin depolymerisation, significant shedding of the glycocalyx from the luminal surface of the cell is observed. This happens together with the loss of focal adhesions on the basal membrane. Using a custom designed electric field (EF) chamber, I demonstrate that the cell migration speed increases by 30~40% following 5h of EF exposure. Cells also show preferred movement towards the anode. However, both are abolished after the enzymatic removal of the glycocalyx, indicating that the speedup and the directional cell migration in applied EF require the presence of the glycocalyx. Even distribution of the glycocalyx on the cell surface at the end of the EF stimulation suggests that EF-directed cell migration is not related to the polarization of the glycocalyx on the cell membrane. All these findings provide a better understanding of the glycocalyx, which will help to develop new strategies for protection of the glycocalyx, restoration of endothelial functions and finally prevention of cardiovascular diseases.

The purpose of this thesis was to examine the interaction between cardiovascular risk factors (particularly novel inflammatory measures) and short-term changes in physical activity. This is important as it is necessary to establish whether the changes that occur to these markers over the longer-term might be a consequence of short-term changes in physical activity. Chapter 4 investigated the challenges in handling the large volume of minute-by-minute data obtained from the use of a novel device for estimating physical activity energy expenditure from synchronous heart rate and accelerometer data. This chapter describes the development of specific software to enable efficient data-processing and evaluated the advantages and disadvantages of this new method of physical activity measurement. Chapter 5 sought to understand the reproducibility of various measures that were central to progress in this field in order to justify their inclusion in future intervention-based studies. This work showed that the inflammatory markers C-Reactive Protein and interleukin-6 and the lipid markers total cholesterol, high-density lipoprotein, low-density lipoprotein and triglycerides were all reproducible measures. The measurement of physical activity energy expenditure, when demarked into common categories, was also mostly reproducible. The circulating marker oxidised-low-density lipoprotein, an in-house adhesion assay and in-house mononuclear cell cytokine secretion assay were determined to be not reproducible and were not used further in this thesis. In Chapter 6 a group of highly active middle-aged men undertook one week of detraining where all structured exercise was removed but activities of daily living were allowed. It was shown that this short-term period of detraining did not elicit any changes in any of the inflammatory, lipid or glucose/insulin markers measured including a commercial, externally-validated whole-blood cytokine secretion assay. In Chapter 7 a sedentary group of middle-aged men performed daily brisk walking for 30 minutes over one week. This period of training did not elicit any change in any of the inflammatory, lipid or glucose/insulin markers measured; including no changes in glucose measures with an oral glucose tolerance test either one day after the last training session or three days later. The differences between the highly-active (Chapter 6) and sedentary (Chapter 7) participants in inflammatory markers were large with substantially higher concentrations for C-Reactive Protein and interleukin-6 in the sedentary middle-aged men. Because these do not change in response to relatively short-term detraining (Chapter 6) or training (Chapter 7) it appears that these differences represent long-term changes and adaptations. Therefore, in addition to being reproducible, fasting inflammatory and lipid markers are very stable with no changes after positive or negative short-term alterations in physical activity level. One immediate implication of this stability is improved ease of follow-up measures after interventions (e.g., training studies) since differences appear to reflect chronic changes in response to the regular training/detraining undertaken and not to recent exercise per se. In the future it will be important to establish better demarcations of acceptable physical activity behaviour. It will also be important to establish whether recently-trained individuals also exhibit stability in their inflammatory markers after a short-period of detraining and whether sedentary individuals are ever capable of achieving the blood profiles of their highly-active counterparts.

The MEF2 transcription factor is a central regulator of skeletal and cardiac muscle development. Recently, we showed that MEF2A regulates skeletal muscle regeneration through direct regulation of the Gtl2-Dio3 microRNA mega-cluster. In addition to their expression in skeletal muscle, temporal expression analysis of selected Gtl2-Dio3 microRNAs revealed high enrichment in cardiac muscle. Therefore, I investigated the role of selected microRNAs from the Gtl2-Dio3 noncoding RNA locus in the heart. First, I found that Gtl2-Dio3 microRNAs are expressed at higher levels in perinatal hearts compared to adult, suggesting they function in cardiac maturation shortly after birth. I also demonstrated that these microRNAs are dependent on MEF2A in the perinatal heart and in neonatal cardiomyocytes. To determine the specific role in cardiac muscle, I overexpressed selected microRNA mimics in neonatal rat ventricular myocytes (NRVMs). My results showed that miR-410 and miR-495 stimulate cell cycle re-entry and proliferation of terminally differentiated NRVMs. Subsequent target prediction analyses revealed a number of candidate target genes known to function in the cell cycle and/or in cardiac muscle. One of these was Cited2, a cofactor required for proper cardiac development. Subsequently, I showed that Cited2 is a direct target of these miRNAs and that siRNA knockdown of Cited2 in NRVMs resulted in robust cardiomyocyte proliferation. This phenotype was associated with reduced expression of Cdkn1c/p57/Kip2, a cell cycle inhibitor, and increased expression of Vegfa, a growth factor with proliferation-promoting effects. Given the exciting possibility of manipulating the expression of these microRNAs to repair the damaged heart by stimulating cardiomyocyte proliferation, I then investigated whether they were regulated in cardiac disease and function in pathological signaling. Toward this end, I examined expression of miR-410, miR-495, miR-433, as well as the Gtl2 lncRNA in various cardiomyopathies. Interestingly, the microRNAs and lncRNA were dynamically regulated in mouse models of cardiac disease including myocardial infarction and chronic angiotensin II stimulation. Furthermore, I showed for the first time that the Gtl2 lncRNA and miRNAs are differentially regulated in myocardial infarction, indicating that the complex regulation of the Gtl2-Dio3 noncoding RNA locus may be important for response to cardiac injury. Lastly, I showed that inhibiting select Gtl2-Dio3 microRNAs in pathological signaling attenuated cardiomyocyte hypertrophy in vitro. Therefore, differential targeting of the Gtl2-Dio3 noncoding RNAs could provide new therapeutic strategies to control the response of the heart to cardiac diseases with diverse etiologies.

A strong body of evidence suggests that environmental insults from the point of fertilisation to birth and neonatal life can shape the health of the individual for many years to come. Adverse exposures, such as maternal overnutrition, in the early life environment increase the risk of traditionally adult-onset diseases such as cardiovascular disease and type 2 diabetes adding greatly to the next generation's burden of disease. Studies in animal models provide strong evidence that these effects are mediated by non-genetic programmed mechanisms. This is of particular concern, as recent studies in the UK suggest that over half of women are now overweight or obese during pregnancy. Current preventative strategies for adult cardiovascular disease have, thus far, focused on reducing an individual's modifiable risk factors. However, given growing evidence that risk of cardiovascular disease is determined in utero, there is strong rationale that disease risk from mother to child could be reduced prior to birth, through targeted interventions in the mother before and during pregnancy. Using an established murine model of maternal diet-induced obesity during pregnancy, the first aim of this thesis was to characterise potential programming factors in the obese mother and identify those that were targeted by a treadmill exercise intervention. Through feeding of an obesogenic diet, dams became heavier, with increased fat mass, and showed insulin resistance at weaning. Previous work has shown the intervention improved maternal insulin sensitivity during pregnancy (E19) and data from this thesis revealed that this was not accompanied by any changes to body composition. Previous data using this model showed that male offspring born to obese dams have pathological cardiac hypertrophy and ex vivo cardiac dysfunction. A second aim of this thesis was to establish if exercise intervention in obese dams was protective to the cardiovascular health of the offspring. These studies revealed that maternal exercise intervention during obese pregnancy had a positive impact by preventing pathological left ventricular cardiac hypertrophy and in vivo dysfunction, but did not prevent programmed hypertension in the male offspring. This demonstrates that offspring cardiac hypertrophy and dysfunction can be programmed independently of hypertension by maternal diet-induced obesity. The third aim of this thesis was to establish how female offspring were impacted by maternal obesity. The results demonstrated that female offspring born to obese dams were hypertensive and displayed right ventricular cardiac hypertrophy. However, there was no observable effect of maternal obesity on cardiac function in female offspring at this age. This highlights the potential sexually dimorphic effects of developmental programming by maternal obesity. A final aim of this thesis was to assess the immediate consequences of maternal obesity on the fetal heart and whether maternal exercise had any impact. This showed that in late gestation (E19), cardiac remodelling were already present in the male fetuses of obese dams, and the exercise intervention did not fully prevent this adverse finding. In conclusion, this thesis highlights that the cardiovascular health legacy of an individual is determined by maternal nutrition before birth and by the intrauterine environment. Just a small improvement in offspring risk could have important implications for the future prevalence of cardiovascular disease worldwide. Importantly this thesis highlights a potential need for combination intervention strategies to tackle the epidemic of obesity in pregnancy, as maternal exercise alone was not sufficient to reduce all aspects of the future burden of cardiovascular disease.

C-type natriuretic peptide (CNP) is synthesised and released by the endothelium and plays a vital role in the maintenance of vascular homeostasis (Moyes et al., 2014). However, a similar regulatory role of endogenous CNP in the heart has yet to be elucidated. Therefore, I have used three unique mouse strains with endothelium (Tie2-Cre), cardiomyocyte (αMHC-Cre) and fibroblast (Col1α2-Cre)-restricted deletion of CNP to investigate if the peptide modulates coronary vascular reactivity and cardiac function. Methods: Langendorff isolated hearts were used to investigate the effect of CNP deletion on coronary vascular reactivity in response to the endothelium-dependent vasodilators bradykinin (10nmol) and acetylcholine (0.1-1nmol). Vasodilatation associated with reperfusion was investigated by transient cessation of flow (20-80 seconds). Ischaemia reperfusion (IR) injury (35 minutes ischaemia followed by 60 minutes reperfusion) was also investigated in cell-specific knockout (KO) animals. Isoprenaline (ISO; 20mg/kg/day, 7days)- and pressure overload (abdominal aortic constriction [AAC]; 6 weeks)-induced heart failure were used to study the effect of CNP deletion during cardiac stress, with cardiac function assessed by echocardiography. Cardiac fibrosis and hypertrophy were determined by picro-sirius red and wheat-germ agglutinin fluorescence staining, respectively. A subset of experiments was repeated in mice with global deletion of natriuretic peptide receptor-C (NPR-C) to delineate the signalling pathway triggered by CNP. Real time qPCR was used to determine hypertrophic and fibrotic gene expression in left ventricles isolated from mice subjected to AAC or sham. Neonatal cardiomyocytes were isolated to investigate angiotensin (Ang)II-induced hypertrophy. Results: Coronary endothelial reactivity was reduced in endothelial CNP (ecCNP) KO mice compared to wild type (WT) in response to bradykinin, acetylcholine and reperfusion-induced vasodilatation. These observations were paralleled in NPR-C KO animals. ecCNP KO did not exacerbate IR injury, whilst mice with cardiomyocyte-restricted deletion of CNP (cmCNP KO) and NPR-C KO animals exhibited a larger infarct size compared to WT. cmCNP KO mice also displayed greater cardiac dysfunction and fibrosis after ISO infusion or AAC compared to WT; similar results were observed in fbCNP KO and NPR-C KO animals. Infusion of CNP (0.2mg/kg/day; osmotic mini-pump, s.c.) in WT, but not NPR-C KO, animals rescued the decline in cardiac function. CNP (1μM) administration in isolated cardiomyocyte also blunted Ang II-induced hypertrophy. Pro-hypertrophic and pro-fibrotic gene expression (ANP, β-MHC and MMP-2) was augmented in cmCNP KO and NPR-C KO mice compared to littermate controls following AAC. Conclusions: Endothelial, cardiomyocyte and fibroblast-derived CNP have distinct, complementary roles in the heart, modulating cardiac function by influencing coronary vascular tone and protecting against heart failure and IR injury. These protective effects of CNP are mediated, at least in part, via NPR-C activation. Developing CNP mimetics or selective NPR-C agonists could be a novel therapeutic intervention in cardiovascular disease.

BACKGROUND: Homeless individuals have higher rates of cardiovascular disease when compared to the general population. Educational curricula in such communities have the potential to decrease the morbidity and mortality associated with hypertension and hypercholesterolemia. However, lifestyle changes from these educational sessions can be difficult for homeless individuals to implement because of competing priorities. OBJECTIVES: The objectives of the “Corazones Sanos” community project are to increase participant knowledge of hypertension and hypercholesterolemia and to counsel individuals on barriers to implementation of therapeutic lifestyle changes in the homeless population. Additional objectives include providing reinforcing support for implementation of lifestyle changes and creation of educational materials related to cardiovascular risk and lifestyle changes for the homeless population. METHODS: This community health education project was implemented with 66 patients from an urban transitional housing facility for homeless individuals recovering from substance abuse. Spanish-speaking patients >18 years old with unstable or lack of housing were eligible to participate in this study. Fifteen minute educational sessions were led by the project coordinator with the end knowledge goal of normal blood pressure and cholesterol values, daily exercise requirement, importance of medication compliance, and healthy diet practices. A reflective approach was utilized to assess participant quotations and themes of barriers to heart healthy living in the homeless population. RESULTS: Participants gained a better knowledge of cardiovascular disease risk factors after participating in education sessions. The main barriers to therapeutic lifestyle changes for prevention of cardiovascular disease included decreased access to healthy foods and fitness opportunities, mental health challenges, lack of social support, and recovering from addiction. About 30% of participants completed a follow-up individual education session. CONCLUSIONS: Casa Esperanza community members gained a better understanding of normal blood pressure and cholesterol ranges, healthy dietary practices, daily exercise requirement, and the importance of medication compliance through this education program. The findings from this project should assist healthcare clinicians with providing effective education for the homeless population to overcome the barriers identified and implement therapeutic lifestyle changes.