The formation of atherosclerotic plaques is linked to a change in vascular function, with evidence of endothelial dysfunction and the proliferation of the underlying vascular smooth muscle cells (VSMCs). Prior to plaque development, risk factors are present that are capable of altering vascular function and promoting disease progression. These risk factors include hypercholesterolaemia, obesity and inflammation. The specific mechanisms of these risk factors in the early stages of atherosclerotic disease development have yet to be fully explored and are likely to be closely interwoven. The aim of this thesis was to assess the effects of these atherosclerotic risk factors, with a primary focus on the direct action of hypercholesterolaemia, on vascular function prior to the development of overt atherosclerosis. After acute ex vivo cholesterol depletion and enrichment, a range of contractile and relaxant stimuli were applied to thoracic aortic rings of wildtype C57BL/6 mice. Cholesterol depletion significantly reduced contractility to phenylephrine (p<0.05) and serotonin (p<0.01). Acute cholesterol enrichment had no effect on vascular contractility, however, acetylcholine stimulated endothelial-dependent relaxation was significantly reduced (p<0.05).Feeding with either a standard chow or a high fat ‘western’ diet was undertaken for eight weeks in both ApoE-/- and C57BL/6 mice. The extent of atherosclerotic disease development was measured through en face lipid staining and histological analysis of aortae. Atherosclerosis was present in the aortic root and intercostal branches of chow and high fat fed ApoE-/- mice but not in diet-matched C57BL/6 mice. No atherosclerotic lesions were observed in the thoracic aortae. In addition, to allow the possibility for direct associations to be made between the associated risk factors of hypercholesterolaemia, obesity and inflammation, and vascular function, a phenotypic assessment of these characteristics was conducted. Wire myography was employed to assess the vascular function of thoracic aortic rings from chow and high fat diet fed ApoE-/- mice and their age and diet matched wildtype C57BL/6 controls. It was found that contractility to both phenylephrine and serotonin was significantly increased in chow fed ApoE-/- mice (both p<0.05). Further investigation into the mechanism, using intracellular calcium imaging and the indo-1 dye, concluded that VSMC store-operated calcium entry was not altered. The exact mechanism behind this increase in contractility is therefore still unknown and there was no clear relationship to the atherosclerotic risk factors assessed. In addition to altered contractility, endothelial-dependent relaxation was shown to be significantly enhanced in high fat fed C57BL/6 (p<0.01) and ApoE-/- mice (p<0.001). Enhanced endothelial-dependent relaxations were transient and sensitive to specific inhibition of cyclooxygenase-2 but not nitric oxide synthase. These changes were hypercholesterolaemia-independent but correlated with signs of obesity and inflammation. In summary, this investigation has demonstrated that vascular function was altered in the murine thoracic aorta prior to overt atherosclerotic plaque development. The implications for these observations relate to the possibility of masked early signs of vascular dysfunction and also the induction of compensatory mechanisms which may have amplified effects over a longer time course; possibly promoting the development and advancement of atherosclerotic disease.

In recent years there has been an explosion in the rates of obesity, defined as a body mass index greater than 30kg/ m2, and associated cardiovascular disease. Alterations in peripheral glucocorticoid metabolism have been suggested to play a role in the development of obesity. Obesity occurs in both sexes, but the risk of associated metabolic disturbance and vascular dysfunction is greater in men. Although there is no accepted definition of obesity in rodents, the term is used to describe animals with a significant increase in fat pad mass often achieved by feeding a high fat diet. Although animal models of obesity have been useful in delineating potential mechanisms linking obesity with its metabolic and vascular sequelae, most studies have been in male animals and, thus, have not addressed sex differences. Additionally, emerging evidence shows that obesity during pregnancy is associated with increased cardio-metabolic and vascular disease in offspring, although the processes underlying such ‘programming’ effects are unclear. This thesis addresses the hypothesis that exposure to postnatal, or maternal obesity will alter both metabolism and vascular function in mice. Male and female mice maintained on a high fat and sugar diet from 5 weeks of age had increased adipose tissue deposition in adulthood. However there were striking sex differences in glucose homeostasis, mRNA levels and glucocorticoid metabolism, with males being more severely affected. Treatment of male mice with 17β-estradiol ameliorated a number of the effects of the high fat diet, including weight gain and altered glucose homeostasis; additionally estradiol altered glucocorticoid metabolism in the adipose so that it resembled that of females. Suprisingly, given the changes in metabolism, obesity in adult mice produced only small changes in vascular function and did not alter vascular remodelling following injury. The effects of maternal obesity were studied using male offspring aged 3 and 6 months. The offspring of obese mothers had similar body weight, adiposity, plasma lipid and plasma hormone concentrations to controls. In contrast, exposure to obesity in utero was associated with receptor specific changes in agonist-mediated contraction and decreased endothelium-dependent relaxation in male offspring. Despite these changes in vascular function, no alterations in blood pressure or vascular remodelling following injury were present. These results demonstrate that the more profound changes in glucose-insulin homeostasis associated with obesity in male humans can be recapitulated in rodent models and imply that estradiol plays a role in protecting the metabolism of female mice, potentially by alteration of glucocorticoid metabolism. Despite altered metabolism in postnatal obesity vascular function remained normal suggesting metabolic and vascular dysfunction are not intrinsically linked. Conversely, maternal obesity did not cause any overt changes in offspring metabolism but caused vascular dysfunction implying these parameters can be programmed independently.

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.

Prospective epidemiological studies indicate that regular exercise during the year prior to conception reduces preeclampsia risk, whereas exercise during affects pregnancy reduces preeclampsia risk only at specific dosages, or in specific subpopulations. The risk of severe preeclampsia is increased among women who exercise for more than 270 minutes/week in early pregnancy. Physiology studies are needed to identify mechanisms through which regular exercise may influence preeclampsia risk. This dissertation examined the effects of pregnancy (30-36 weeks gestation), and regular exercise participation, on two important pathophysiological features of preeclampsia; circulating anti-angiogenic markers, represented by soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sEng), and endothelial dysfunction. The results demonstrate that regularly exercising, pregnant non-smoking women have higher levels of serum placental growth factor (PlGF), lower levels of serum sFlt-1 and sFlt-1:PlGF, and are less likely to experience high serum sEng levels, than sedentary women. The effects of exercise on PlGF and sFlt-1:PlGF are more pronounced among women exercising less than 270 minutes/week in pregnancy. Anti-angiogenic changes that could contribute to preeclampsia were not observed immediately after short-duration, moderate-intensity exercise in the third trimester. Flow-mediated dilation (FMD) and the shear stimulus for FMD are not affected by pregnancy, however the time to peak FMD was increased in pregnancy. Regular exercise did not affect FMD or its shear stimulus in healthy pregnant or non-pregnant women. FMD and its shear stimulus were positively correlated in active, but not inactive, pregnant and non-pregnant women. Pregnancy and physical activity do not affect radial artery low flow-mediated constriction (L-FMC). L-FMC is artery dependent, occurring in the radial, but not the brachial, artery of healthy pregnant and non-pregnant women. The positive correlation between L-FMC and FMD suggests that L-FMC and FMD are not independent measurements. The results of this thesis suggest that physical activity and exercise may reduce preeclampsia risk by reducing concentrations of angiogenic markers. Although exercise participation did not affect conduit artery vascular function in healthy pregnant women, future studies should investigate the effects of exercise on vascular function in women with endothelial dysfunction, or risk factors for preeclampsia. / Thesis (Ph.D, Kinesiology & Health Studies) -- Queen's University, 2009-06-11 12:41:53.466

High consumption of dairy products, particularly non-fat dairy, is associated with reduced risk of high blood pressure and vascular dysfunction. Currently, it is not known if the solitary addition of conventional non-fat dairy products to the normal routine diet is capable of reducing blood pressure or improving vascular function. Accordingly, the primary aims of the present study were to determine if the solitary addition of conventional non-fat dairy products to the normal routine diet would reduce blood pressure and improve vascular function in middle-aged and older adults with elevated blood pressure. Using a randomized, crossover intervention study design, forty-nine adults with elevated blood pressure underwent a High Dairy condition (+4 servings/day of conventional non-fat dairy products) and isocaloric No Dairy condition (+4 servings/day fruit products) in which all dairy products were removed. Both dietary conditions lasted 4 weeks with a 2-week washout before crossing over into the alternate condition. In Study 1, the High Dairy condition produced reductions in brachial systolic blood pressure and pulse pressure. The hypotensive effects were observed within three weeks after the initiation of dietary intervention and in both casual seated and ambulatory (24-hour) measurements. On the contrary, pulse pressure was increased after removal of all dairy products in the No Dairy condition compared to baseline and after in the High Dairy condition. There were no changes in diastolic blood pressure after either dietary condition. In Study 2, the High Dairy condition produced reductions in carotid systolic blood pressure, pulse pressure, and carotid-femoral pulse wave velocity with a concomitant increase in brachial flow-mediated dilation and cardiovagal baroreflex sensitivity. Brachial flow-mediated dilation decreased and carotid pulse pressure increased after removal of all dairy products in the No Dairy condition. Furthermore, [delta] carotid systolic blood pressure and carotid-femoral pulse wave velocity were highly related. Taken together, we concluded that the solitary manipulation of conventional dairy products, particularly non-fat dairy, in the normal routine diet would modulate levels of blood pressure and vascular function in middle-aged and older adults with pre-hypertension and hypertension. / text

Neointimal hyperplasia, the proliferative growth of the innermost layer of the blood vessel wall, is a key process in the response to vascular injury, underlying conditions such as post-interventional restenosis and vein/arterial graft disease. One of the many mediators implicated in this process is endothelin-1 (ET-1), a potent vasoconstrictor with pro-inflammatory and pro-mitogenic actions, which acts through ETA and ETB receptor subtypes. It is well established that ET-1 increases, and ETA blockade reduces, neointima formation following vascular injury. The role of ETB is less clear because these receptors mediate potentially beneficial actions in endothelial cells (EC; such as nitric oxide production, and ET-1 clearance) but detrimental effects elsewhere (such as vascular smooth muscle) and it has been recently reported that non-cell-specific ETB deficiency is associated with increased neointimal lesion size following injury. The work described in this thesis addressed the hypothesis that endogenous ET-1 contributes to neointimal hyperplasia by activation of the ETA receptor, and that this action is moderated by concurrent activation of the ETB receptor expressed in EC. The role of ET receptors in neointimal lesion development was assessed using two models of femoral arterial injury in the mouse: (i) an established method of intraluminal wire-injury, and (ii) adaptation of a model of ligation injury that induces robust neointimal lesion formation without physical damage to the endothelium. Lesion development was assessed using standard histological techniques and this was augmented by development of quantitative optical projection tomography (OPT) to allow three-dimensional analysis of lesions. The role of ETA and ETB receptors in these models was addressed using suitable pharmacological ET receptor antagonists. Following wire-injury, selective ETB blockade (A192621; 30mg.kg-1.day-1; 35 days) increased lesion size and blood pressure without significant altering lesion composition. In contrast, selective ETA blockade (atrasentan; 10mg.kg-1.day-1; 35 days) reduced lesion size and blood pressure. Combined ETA+ETB antagonism had no effect on lesion size, despite reducing blood pressure, and reducing collagen content of the lesions. In the ligation model, neither ETA selective, ETB selective nor ETA+ETB blockade altered lesion size as assessed by standard histology but analysis by OPT indicated that ETA blockade, with or without concurrent ETB blockade, reduced lesion volume. The influence of ETB receptors expressed by ECs on lesion formation was addressed using EC-specific ETB knockout mice. Small vessel myography indicated that endothelium-dependent relaxation was unaltered in femoral arteries from these mice. In addition, no effect on lesion size or rate of development was observed in either wire- or ligation-injury models of neointima formation (although subtle effects on lesion and medial composition were apparent after intra-luminal injury). These results indicate that ETB receptor activation can moderate the detrimental actions of the ETA receptor on neointimal lesion progression, and that this role is dependent on the mode of vascular injury. Furthermore, in this setting, this beneficial action is not primarily mediated by ETB expressed by EC, suggesting that ETB in other cell types can reduce lesion development through another, unidentified mechanism. Therefore, while both ETA selective and non-selective ETA/B antagonists are currently in clinical use, in conditions where similar arterial remodelling processes occur, selective ETA receptor antagonists might be preferred.

One of the earliest sub-clinical stages associated with atherosclerosis is endothelial dysfunction (ED), which has been shown to predict future cardiovascular events. Chronic exercise is thought to improve endothelium-dependent vasodilation; however, few studies have evaluated the effects of acute exercise on vascular function (VF). Moreover, studies evaluating ED following an exercise training program lack a standardized time frame in which to measure VF. Although most studies require subjects to abstain from exercise for 24 hours prior to any VF measure, no study to date has assessed VF longer than 24 hours after the cessation of exercise. Additionally, no studies have compared VF responses in obese and non-obese individuals following acute exercise. Purpose: Therefore, the purpose of this study was to evaluate VF, as determined by the assessment of forearm blood flow (FBF) and vascular reactivity (VR) before and up to 48 hours after a single bout of maximal exercise in obese and non-obese males. Methods: Twelve obese (37.0 ± 1.1 kg/m2) and twelve non-obese (21.9 ± 0.3 kg/m2) males volunteered to participate. FBF was assessed before and during reactive hyperemia (RH). FBF measures were obtained prior to (PRE-E), immediately after (POST-E), and at 1 (POST-1), 2 (POST-2), 24 (POST-24), and 48 (POST-48) hours after exercise. Total excess flow, calculated as the difference between baseline FBF and FBF during RH, was used as an indicator of VR. Blood samples were also obtained at each time point to evaluate the response of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α), which are potential modifiers of VF. Results: Baseline FBF and FBF during RH were significantly (P < 0.05) increased in both groups POST-E before returning to baseline values by POST-1. VR was enhanced in both groups POST-E, although the magnitude of change was greater in non-obese males. VR was significantly (P < 0.05) increased in non-obese males POST-E and was not significantly (P < 0.05) reduced until POST-48. Concentrations of IL-6 and TNF-α were unchanged in response to exercise in non-obese and obese males. Conclusions: An acute bout of maximal exercise significantly increased forearm endothelium-dependent vasodilation in non-obese and obese males. Additionally, an increased reactive vasodilation was observed only in non-obese males following exercise. These results also suggest that in non-obese males, measurements used to verify improvements in VF following exercise training should be employed after a minimum of 48 hours following physical activity.

Objectives: 1) Evaluate the risk of atherosclerosis in rheumatic disease compared to healthy controls; 2) Assess the lipid profile of children with systemic lupus erythematosus (SLE) at presentation before treatment with corticosteroids; 3) Compare the lipid profiles of children with juvenile dermatomyositis (JDM), systemic juvenile idiopathic arthritis (SJIA), and SLE; 4) Evaluate the extent of early atherosclerosis in children with JDM, SJIA, and SLE; 5) Investigate the progression of early markers of atherosclerosis in children with SLE. Methods. The methods include a systematic review, a cross sectional study of serum lipid levels of a cohort of children with SLE, an analysis of the first time point of a prospective study of cardiovascular disease risk factors and vascular function measures of a cohort of children with JDM, and SJIA, and SLE and a longitudinal study of vascular function measures of a prospective study of a cohort of children with SLE. Results. Our systematic review demonstrated that carotid intima media thickness (CIMT), a surrogate marker of early atherosclerosis, was significantly increased in rheumatic disease populations. We found that newly diagnosed children with SLE before corticosteroid treatment exhibited a pattern of dyslipoproteinemia of increased triglycerides and depressed HDL-cholesterol. When we measured the lipid profiles in children with the rheumatic diseases of JDM, SJIA, and SLE, one third of children had at least one abnormal lipid value. The most common abnormalities were found for total cholesterol and triglyceride levels and most often in children with JDM. One quarter of all patients were found to have insulin resistance. Lastly, when we considered the effects of treatment in children with SLE, we found that improvement in CIMT was possible and it correlated with a higher cumulative dose of prednisone over the study period. Conclusions. Early markers of atherosclerosis in pediatric rheumatic disease are important for determining the risk of these children in developing heart disease as young adults. Chronic inflammation plays a significant role and should be considered an important predictor of premature atherosclerosis.

Objectives: 1) Evaluate the risk of atherosclerosis in rheumatic disease compared to healthy controls; 2) Assess the lipid profile of children with systemic lupus erythematosus (SLE) at presentation before treatment with corticosteroids; 3) Compare the lipid profiles of children with juvenile dermatomyositis (JDM), systemic juvenile idiopathic arthritis (SJIA), and SLE; 4) Evaluate the extent of early atherosclerosis in children with JDM, SJIA, and SLE; 5) Investigate the progression of early markers of atherosclerosis in children with SLE. Methods. The methods include a systematic review, a cross sectional study of serum lipid levels of a cohort of children with SLE, an analysis of the first time point of a prospective study of cardiovascular disease risk factors and vascular function measures of a cohort of children with JDM, and SJIA, and SLE and a longitudinal study of vascular function measures of a prospective study of a cohort of children with SLE. Results. Our systematic review demonstrated that carotid intima media thickness (CIMT), a surrogate marker of early atherosclerosis, was significantly increased in rheumatic disease populations. We found that newly diagnosed children with SLE before corticosteroid treatment exhibited a pattern of dyslipoproteinemia of increased triglycerides and depressed HDL-cholesterol. When we measured the lipid profiles in children with the rheumatic diseases of JDM, SJIA, and SLE, one third of children had at least one abnormal lipid value. The most common abnormalities were found for total cholesterol and triglyceride levels and most often in children with JDM. One quarter of all patients were found to have insulin resistance. Lastly, when we considered the effects of treatment in children with SLE, we found that improvement in CIMT was possible and it correlated with a higher cumulative dose of prednisone over the study period. Conclusions. Early markers of atherosclerosis in pediatric rheumatic disease are important for determining the risk of these children in developing heart disease as young adults. Chronic inflammation plays a significant role and should be considered an important predictor of premature atherosclerosis.

There is an increasing recognition that vascular disease risk is associated with a greater incidence of cognitive impairment and dementia. Such link is supported by the physiological observation that cerebral metabolism heavily relies on vascular supply of oxygen and energy substrates. Cerebral hypoperfusion which results from vascular dysfunction causes a mismatch between energy demand and supply and is associated with the pathological features of dementia, including the impairments of action potential generation and protein synthesis, glutamatergic excitotoxicity, and the deposition of cerebral amyloid-β proteins. In contrast, habitual aerobic exercise is an established strategy to ameliorate the risk factors for vascular disease and is increasingly recognized in improving cognitive function. Accordingly, the primary purpose of this dissertation study was to investigate whether the exercise-related improvement in cognitive function was attributable to ameliorated vascular function and risk factors for vascular disease. In order to address this as comprehensively as we could, both cross-sectional and interventional studies were conducted. The primary findings from the present study were as follows. In the cross-sectional study, a greater cognitive performance observed in endurance-trained adults was associated with higher levels of cerebral CO2 reactivity and brachial endothelium-dependent vasodilation and lower levels of central arterial stiffness and plasma insulin. In the interventional study, a 3-month aerobic exercise training intervention did not improve cognitive function although central arterial stiffness and brachial endothelium-dependent vasodilation made favorable changes. However, we found that the improvement in memory performance after aerobic exercise training was associated with the reduction in central systolic blood pressure. Taken together, a better cognitive performance observed in endurance-trained adults may not directly be attributable to greater vascular function because there were discrepant changes in cognitive and vascular functions after a 3-month aerobic exercise intervention. The correlation between the changes in memory performance and central systolic blood pressure is interesting but needs further investigation using a larger sample size. The discrepancy in the results between the cross-sectional and interventional studies could be explained by the duration of exercise training and/or the time it takes for the effect of improved vascular function to translate into cognitive function. / text