Reabsorbing ~7% of the sodium load, the distal convoluted tubule plays key roles in blood pressure (BP) homeostasis. Here, Na-Cl co-transport (NCC) is the major route for apical Na entry making thiazide diuretics (NCC inhibitors) a mainstay hypertension treatment. Predictive adaptations of sodium excretory rhythms are supported by an intrinsic renal clock, which regulates transporter activity according to physiological need. Peripheral clocks can be influenced by glucocorticoids, which also have a circadian rhythm. We therefore hypothesized that NCC’s diurnal rhythm is regulated by glucocorticoids. C57BL6 mice were kept on a 12h light cycle with subjective day starting at 7am. Urine was collected in 12h periods and kidneys harvested at 1am (night) and 1pm (day). Slc12a3 (NCC encoding gene) mRNA and NCC protein abundance were similar between day and night but NCC phosphorylation at threonine 53 was significantly higher at night compared to day. Plasma and urinary corticosterone levels were elevated at night. Glucocorticoid inducible leucine zipper (GILZ) and serum and glucocorticoid inducible kinase (SGK1) transcripts also increased at night. Chronic corticosterone infusion flattened the plasma corticosterone rhythm within an intermediate physiological range. The diurnal rhythm of pT53 NCC was dampened in these mice but not in vehicle-treated mice. Blood pressure was monitored in the mice by radiotelemetry. After 2 weeks of baseline measurements mice received chronic corticosterone or vehicle for 3weeks, during the last 10 days of which they received ~80 mg/kg hydrochlorothiazide in their drinking water. At night systolic BP (SBP) was unaffected by any treatment whereas during the day SBP significantly increased following corticosterone but was unaffected by vehicle. Cosinor analysis of SBP in corticosterone and vehicle treated mice showed a marked reduction in rhythmicity, increased MESOR and reduced amplitude. In animals receiving corticosterone HCTZ partially rescued daytime SBP. This manoeuvre also improved SBP rhythmicity, reduced MESOR and increased amplitude. These data indicate that NCC phosphorylation has a diurnal rhythm that is in part regulated by glucocorticoids. They also show that alteration of the glucocorticoid rhythm affects the blood pressure rhythm in part through its effect on NCC phosphorylation. These findings may be clinically relevant in the pathogenesis of hypertension in conditions associated with elevated glucocorticoid levels such as metabolic syndrome and chronic stress.

Thesis (M.S.)--Boston University

OBJECTIVE: While it has long been known that vascular endothelial growth factor A (VEGF-A) plays a role in vascular homeostasis, only recently have its effects been explored in adipose tissue. As perivascular adipose tissue (PVAT) is in close proximity with the aorta and coronary arteries and is known to contribute to vasodilation, it may influence vascular function via secretion of VEGF-A. The objective of this study is to analyze the effects of brown-adipocyte deletion of VEGF-A on circulating VEGF-A levels and distribution of VEGF-A isoforms. We hypothesize that ablation of VEGF-A in brown adipocytes will affect perivascular adipocyte and vascular function. MATERIALS/ METHODS: Mice harboring a brown adipose-specific VEGF deficiency, UCP1cre.VEGFflox/flox mice, were maintained on a chow diet. Primary adipocytes were isolated from brown adipose tissue (BAT) and thoracic PVAT by collagenase digestion and culturing. Gene expression was measured by RT-PCR from RNA extracted from tissues of UCP1cre.VEGFflox/flox mice. Circulating and tissue VEGF-A levels were quantified by ELISA. RESULTS: While VEGF-A ablation using the UCP1 promoter decreases VEGF- protein A levels in BAT and PVAT, it does not affect VEGF-A levels in the circulation. CONCLUSION: This study confirms the functional utility of the UCP1cre.VEGFflox/flox mouse model, as it selectively reduces VEGF-A levels in BAT and PVAT without affecting other tissues or circulating levels. As previous studies using VEGF ablation in all adipose tissues demonstrate an impaired thermogenic response and brown-adipocyte dysfunction, further study of the brown adipose-specific mouse model is warranted. Because PVAT provides protection against vascular stiffness, modulation of VEGF-A in PVAT may be a viable treatment for obesity-associated vascular complications.

Cardiovascular disease remains one of the leading causes of mortality globally. Innovative techniques are required to tackle its anticipated rise due to rising obesity, diabetes and an ageing population. Personalised electronic coaching (eb coaching) using the Internet and emails may help motivate healthier living and be of clinical benefit in complementing current programmes for cardiovascular risk reduction. I investigated whether personalised ebcoaching on top of SOC was more clinically effective than SOC alone, in reducing cardiovascular risk in asymptomatic individuals with high cardiovascular risk. I lead a randomised controlled trial of 402 participants using robust surrogate markers to identify change over 6 months. I assessed the feasibility of using cardiovascular magnetic resonance surrogate markers to guide their use in future studies of lifestyle interventions. I performed systematic reviews to identify 1) similarities and differences among leading primary prevention guidelines that address cardiovascular screening and risk assessment and 2) guideline recommendations on lifestyle advice and interventions to identify how ebcoaching could be used and what advice to incorporate in ebcoaching platforms. I found modest but statistically significant improvements in both ebcoaching and SOC groups to a similar level. Personalised ebcoaching did not show additional benefit in a highbrisk primary prevention cohort. It is feasible to use cardiovascular surrogate markers derived from cardiovascular magnetic resonance in lifestyle interventions studies. However, further studies correlating change in these markers with longbterm outcomes are required. Considerable discrepancies exist in the guidelines on risk on cardiovascular screening and risk assessment, with no consensus on optimum screening strategies or classification of high risk thus affecting treatment threshold. Guidelines did highlight the importance of lifestyle interventions in primary prevention and generally provided similar advice. Ebcoaching should not be incorporated into current prevention programmes for high risk populations unless the tools are improved and effectiveness is proven.

Orientador: Antonio Sérgio Martins / Banca: Emilio Carlos Curselli / Banca: Walter José Gomes / Resumo: Apresentamos nesta dissertação, uma revisão dos estudos epidemiológicos e econômicos mostrando a doença cardiovascular como a grande pandemia do século XXI, tanto dos países desenvolvidos como naqueles em desenvolvimento. Colocamos em foco a necessidade de estudar-se a epidemiologia cardiovascular, que consiste na busca das causas de mudança da condição de saúde ou doença, nos seus fatores de risco e nos esforços para a preservação da saúde. Além das perspectivas epidemiológicas, há de se comentar as econômicas e sociais que seguem as primeiras uma vez que pessoas cada vez mais jovens adoecem causando um déficit na população economicamente ativa. Hoje a doença cardiovascular consome muitos recursos, saber quanto custa ao serviço público, com dados claros e fidedignos é a única forma de evitar um caos nos sistemas de saúde de todo o mundo. A grande maioria dos dados disponíveis hoje necessita de aperfeiçoamento para serem utilizados com êxito. No Brasil a criação do Sistema Único de Saúde SUS em 1988 norteada com os princípios da universalidade e da equidade, juntamente com os avanços tecnológicos e escassez de recursos, exige uma otimização dos recursos disponíveis sem, entretanto alterar a qualidade da assistência á saúde. Quando analisamos os gastos do SUS em saúde, observamos um incremento anual crescente, especialmente quando falamos das doenças cardiovasculares. Em 2007 foram registradas 1.157.509 internações por DCV no Sistema Único de Saúde (SUS) que custaram aos cofres públicos R$ 1.468.441.279,46. Em novembro de 2009 ocorreram 91.970 internações por DCV, resultando em um custo de R$ 165.461.644,33 (DATASUS). Os gastos continuam aumentando ano após ano de forma desastrosa. Sendo assim caracterizamos o gasto com as DCV especificamente... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: We presented in this dissertation, a review of epidemiologic and economic studies showing the cardiovascular disease as the great pandemic of the XXI century much of the developed countries as in developing countries. We focuses the need to study the cardiovascular epidemiology, which consists in finding the causes of change in health condition or disease, in its risk factors and efforts to preserve health. Apart from the epidemiological perspective, we should also comment on the economic and social that follows the first since more and more young people become ill, causing a deficit in the economically active population. Today, cardiovascular disease consumes too many resources, how much it costs to public service, with clear and reliable data is the only way to avoid chaos in health systems around the world. The vast majority of data available today need improvement to be used successfully. In Brazil, the creation of the Sistema Único de Saúde in 1988 SUS guided with the principles of universality and equity, along with technological advances and dwindling resources, requires an optimization of available resources without however changing the quality of healthcare. When we analyze SUS expenses on health, we observe a growing annual increase, especially when it comes to cardiovascular diseases. In 2007 we registered 1,157,509 admissions for CVD in the Sistema Único de Saúde (SUS) that cost the public treasury R$ 1,468,441,279.46. In November 2009 there were 91,970 admissions for CVD, resulting in a cost of R$ 165,461,644.33 (DATASUS). Expenses continue to increase year after year in a disastrous way. Therefore we characterize the CVD expenses specifically heart disease by the government through a retrospective study, with descriptive purpose, developed within the specialty of cardiology where numerical data... (Complete abstract click electronic access below) / Mestre

Background: Systemic sclerosis (SSc) is an autoimmune disease associated with significant mortality and morbidity. Cardiovascular causes are the single largest contributor to premature death. To date, much of the focus on managing the care of SSc patients has concentrated on traditional risk factors related to fibrotic and microvascular dysfunction. There is, however, evidence of a strong cardiovascular component to the disease and points to macrovascular dysfunction as being a key contributor to the premature mortality associated with SSc. This thesis reports on the conduct of a multi-centre, randomised, placebo-controlled clinical trial (the SSTEP Study). The aim of the study was to assess whether oral Iloprost was more effective than placebo in reducing cardiovascular events and disease progression in SSc. Methods: Two hundred and sixteen patients with systemic sclerosis were recruited, between February 2002 and February 2005, at nine centres in the UK and Ireland. After one month placebo run-in, participants were randomised to either oral Iloprost (50-200mcg daily) or matched placebo. Baseline demographics, disease characteristics and organ screening data were collected, and participants were reviewed annually for endpoint measurements; CV events, SSc disease progression and mortality, with regular safety reviews between these annual visits. Participants were followed up for a period of 4 to 7 years. Results: Data analysis of the combination of the two measures (survival free from death or a cardiovascular event) demonstrated a trend towards favouring Iloprost over placebo but the difference was not statistically significant (Logrank test: Chi square=0.75, p=0.39). When time to a confirmed cardiovascular endpoint alone was examined there was a suggestion of a benefit from Iloprost, but the difference was again not statistically significant (Logrank test, Chi square =0.82, p=0.37). There was no statistically significant change in the rate at which organ screening endpoints occurred throughout follow-up, and for each endpoint there was no statistically significant difference between results in patients randomised to Iloprost compared to those randomised to placebo. Withdrawal from the treatment to which the patient was randomised was frequent with 97 (45%) of the total participants discontinuing study medication. ‘On treatment’ analysis, undertaken using the endpoint of death or confirmed cardiovascular endpoint, just failed to show statistical significance at the 5% level (p=0.054). Conclusion: The results of the SSTEP study showed that there was a trend towards favouring oral Iloprost over placebo in systemic sclerosis, though there was no statistically significant evidence to recommend its use to prevent disease progression. The high rate of withdrawal from both Iloprost and placebo hindered the possibility of demonstrating that Iloprost was effective in this study. It cannot be concluded that it is a useful therapy that may prevent premature mortality or progression to cardiovascular disease in this patient group.

Cardiovascular diseases (CVD) are united in pathology by atherosclerosis; signal transduction is essential in this process for the expression of cell adhesion molecules early in the disease, capturing, tethering and transmigrating monocytes into the sub-endothelial space. The local recruitment of inflammatory cells and release of pro-inflammatory mediators induces endothelial dysfunction, an early functional abnormality in CVD. Plasma cytokines have been used to stratify CVD risk in humans. Studies have shown associations between pro-inflammatory tumour necrosis factor (TNF) and interleukin-6 (IL-6) and adverse cardiovascular events. However, the early pathophysiological signalling responsible for the expression of inflammatory molecules in vascular dysfunction remains poorly defined with limited in vivo studies. The ability to quantify endothelial dysfunction in animal models is limited by the need to cull animals in order to obtain vascular tissue, prohibiting longitudinal studies. The development of an in vivo non-invasive technique in this thesis has allowed the longitudinal assessment of microvascular responses in mouse models of inflammation. Candidate kinases in innate inflammatory signalling were assessed in vivo to better understand their role in endothelial dysfunction. These proteins are either essential in inflammatory homeostasis (A20 binding inhibitor of NF-ĸB-1 and mitogen and stress activated kinase 1/ 2) through negative regulation of inflammation or are fundamental in the cascade for cytokines synthesis (myeloid differentiation primary response gene 88, mitogen activated protein kinase activated-protein kinase 2/ 3). Through genetic alteration these models 19 produced a hyper-inflammatory CVD prone phenotype or one that is cardiovascular protective, respectively. Endothelium-dependent vasodilatation was attenuated in the presence of dyslipidaemia through reduced of nitric oxide (NO), cholesterol feeding induced increased expression of IL-6, IL-1α and TNF-α. Importantly, through abrogation of cytokine signalling, NO was preserved in the presence of dyslipidaemia through reduced cytokine release of IL-6 and IL-1α. This data provides a novel insight into the cellular signalling in inflammation and subsequent cardiovascular health, with a translational potential to effectively enhance the understanding of clinical pathology and inflammatory risk. These pathways offer unique therapeutic avenues for pharmacological intervention to potentially limit CVD in the human population.

Time with media screens (television, computers, videogames, cell phones, and tablets) is the primary activity of youth, second only to sleeping, and represents a major risk factor for cardiovascular diseases (CVD). Additionally, the populations with highest rates of screen time are also those most at risk of CVD from genetic predisposition (i.e., Blacks, Hispanics). The purpose of this descriptive, correlational study, based on cross-sectional analysis of archived data from the 2009 - 2010 NHANES for United States youth, newborn to 20 years old, was to determine whether the combination of media screen time with genetic background is a better predictor of CVD than either factor alone. The theoretical framework was the social ecological theory of disease distribution. The relationship between media screen time, genetic background, and CVD risk factor was determined using binary logistic regression. Results of this study indicated that the relationship between ethnicity, gender, and type/duration of exposure to media screen is important to predict the CVD risk factors C-reactive protein (CRP), triglycerides, and diastolic blood pressure. Interventions that limit exposure total screen time will reduce the risk of increased blood pressure among all races. However, culturally relevant intervention should be designed specifically for non-Hispanic Blacks, other Hispanics, and other race. These ethnicities have the highest propensity to increase in blood pressure, CRP, and triglycerides and also spend the largest amount of time in front of the media screen. Results from this study may help to promote policies and initiatives to limit screen time that are culturally relevant and more focused.

Endothelial progenitor cells (EPCs) have reduced neovascularization capacity in the context of coronary artery disease (CAD) or cardiac risk factors (RFs). Since, endothelial NO synthase (eNOS) is critical to normal EPC function, we hypothesized that bone marrow cells (BMCs) from rats with RFs and EPCs from humans with CAD and/or RFs show dramatically reduced neovascularization capacity in vitro and in vivo, which can be reversed by eNOS overexpression. BMCs were isolated from rat models of type II diabetes and the metabolic syndrome, and we showed a significant reduction in their ability to stimulate neovascularization in vitro and in vivo. In humans, we isolated circulating ‘early EPCs’ from healthy subjects and patients with CAD and RFs, and transduced them using lentiviral vectors containing either eNOS or GFP (sham). EPCs from patients had reduced in vitro migration in response to SDF-1 or VEGF, which was reversed by eNOS-transduction. In co-culture with human umbilical vein endothelial cells (HUVECs) on Matrigel, eNOS-transduced EPCs contributed to increased and more complex angiogenic tube formation compared to sham-transduced cells. Human EPCs from patients were ineffective in enhancing ischemic hind limb neovascularization and perfusion in a nude mouse, whereas eNOS-transduced EPCs resulted in a significant improvement compared to sham-transduced cells. In a swine model of acute myocardial infarction (MI), eNOS- and non-transfected BMCs both increased left ventricular function compared to sham. However, there was no benefit to eNOS overexpression in this model. Various differences in the models and procedures may explain the incongruous results obtained. Taken together, these results show that eNOS overexpression significantly improves the neovascularization capacity of EPCs of human subjects with CAD and RFs and could represent an effective adjunctive approach for the improvement of autologous cell therapies for cardiovascular disease.

Endogenously, VEGF initiates angiogenesis, then later Angiopoietin (Ang)-1 matures vessels. We hypothesized that multigene therapy of VEGF before Ang1 to ischemic hindlimb tissue would result in persistent angiogenesis. At 2, 4 and 8 wks after inducing ischemia, blood flow was assessed by contrast-enhanced ultrasound. Animals were treated with VEGF at 2 wks, VEGF/Ang1 at 2 wks, or VEGF at 2 wks and Ang1 at 4 wks. In untreated controls, blood flow remained reduced. After VEGF delivery, resting flow and vessel density increased; however, flow reserve remained reduced, and vasculature was capillary-rich and eventually regressed. After VEGF/Ang1 co-delivery, flow increased marginally, flow reserve improved and vascular architecture remained normal. After separated VEGF and Ang1 delivery, flow, vessel density and flow reserve increased and were sustained, while vascular architecture remained normal. In conclusion, temporally separated VEGF and Ang1 delivery promotes sustained angiogenesis and improved vessel functionality.