Master of Science / Department of Kinesiology / Timothy I. Musch / Aged rats exhibit a decreased muscle microvascular O[subscript]2 partial pressure (PO[subcript]2mv) at rest as well as during contractions compared to young rats and this may contribute to their reduced exercise tolerance. Age-related reductions in nitric oxide (NO) bioavailability due, in part, to elevated reactive O[subscript]2 species (ROS) constrain muscle blood flow (Qm). Therefore, antioxidants may restore NO bioavailability, Qm and ameliorate the reduction in PO[subscript]2mv and hence the decrease in exercise tolerance seen in aged rats. PURPOSE: To test the hypothesis that antioxidants would elevate Qm at rest and during contractions and therefore PO[subscript]2mv in aged muscle. METHODS: PO[subscript]2mv and Qm were measured in the spinotrapezius while muscle oxygen consumption (VO[subscript]2m) was estimated in 20 anesthetized male Fisher 344 x Brown Norway hybrid (F344xBN) rats at rest and during 1 Hz contractions before and after antioxidant intravenous infusion (76mg/kg vitamin C and 52mg/kg tempol). Moreover, muscle force production was measured in a subset of animals. RESULTS: Before infusion, contractions invoked a biphasic PO[subscript]2mv that fell from 30.6 [plus or minus] 0.9 mmHg to a nadir of 16.8 [plus or minus] 1.2 mmHg with an 'undershoot' of 2.8 [plus or minus] 0.7 mmHg below the subsequent steady-state (19.7 [plus or minus] 1.2 mmHg). Antioxidants elevated baseline PO[subscript]2mv to 35.7 [plus or minus] 0.8 mmHg (P<0.05) and reduced or abolished the 'undershoot' (P<0.05) without changing the steady-state contracting PO[plus or minus]2mv. Antioxidants did not change Qm at rest but during contractions Qm was reduced from 157 [plus or minus] 28 to 91 [plus or minus] 15 ml min[superscript]-1 100g[superscript]-1 (P<0.05). Antioxidants produced no significant effect on VO[subscript]2m. However, antioxidant supplementation produced a 16.5% decrease (P<0.05) in muscle force production that occurred within the first contraction and remained throughout the duration of stimulation. In addition, the ratio of muscle force production to VO[subscript]2m (F/VO[subscript]2m) actually increased from 0.92 [plus or minus] 0.03 to 1.06 [plus or minus] 0.6 (P<0.05) following infusion of antioxidants. CONCLUSION: Antioxidant supplementation significantly alters the balance between muscle O[subscript]2 delivery and VO[subscript]2 at rest and during contractions, which modifies the microvascular PO[subscript]2mv profile. Specifically, antioxidants elevate PO[subscript]2mv, which improves the potential for diffusive blood-myocyte flux. This effect arises, in part, from the unanticipated fall in muscle force production consequent to antioxidant supplementation.

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The importance of coronary microcirculatory perfusion is highlighted by the severe impact of microvascular diseases such as diabetes and hypertension on heart function. Recently, highly-detailed three-dimensional (3D) data on ex vivo coronary microvascular structure have become available. However, hemodynamic information in individual myocardial capillaries cannot yet be obtained using current in vivo imaging techniques. In this thesis, a novel data-driven modelling framework is developed to predict tissue-scale flow properties from discrete anatomical data, which can in future be used to aid interpretation of coarse-scale perfusion imaging data in healthy and diseased states. Mathematical models are parametrised by the 3D anatomical data set of Lee (2009) from the rat myocardium, and tested using flow measurements in two-dimensional rat mesentery networks. Firstly, algorithmic and statistical tools are developed to separate branching arterioles and venules from mesh-like capillaries, and then to extract geometrical properties of the 3D capillary network. The multi-scale asymptotic homogenisation approach of Shipley and Chapman (2010) is adapted to derive a continuum model of coronary capillary fluid transport incorporating a non-Newtonian viscosity term. Tissue-scale flow is captured by Darcy's Law whose coefficient, the permeability tensor, transmits the volume-averaged capillary-scale flow variations to the tissue-scale equation. This anisotropic permeability tensor is explicitly calculated by solving the capillary-scale fluid mechanics problem on synthetic, stochastically-generated periodic networks parametrised by the geometrical data statistics, and a thorough sensitivity analysis is conducted. Permeability variations across the myocardium are computed by parametrising synthetic networks with transmurally-dependent data statistics, enabling the hypothesis that subendocardial permeability is much higher in diastole to compensate for severely-reduced systolic blood flow to be tested. The continuum Darcy flow model is parametrised by purely structural information to provide tissue-scale perfusion metrics, with the hypothesis that this model is less sensitive and more reliably parametrised than an alternative, estimated discrete network flow solution.

In recent times the potential impact of preeclampsia on the cardiovascular health of offspring has been identified. This thesis explores the relationship between preeclampsia and offspring cardiovascular phenotype during the first three decades of life. A systematic review and meta-analysis provided evidence that there was increased blood pressure and BMI in the offspring of preeclamptic pregnancies (n = 45,249). There was however limited data on metabolic features and inadequate characterisation of the degree of prematurity or growth restriction in existing literature. I therefore studied data on two birth cohorts with up to 28 years of detailed prospective follow up (n = 2868 and n = 926). Those born very preterm to preeclamptic pregnancies had transient perinatal reductions in insulin and cholesterol, although extreme prematurity was the only determinant of variation in cardiovascular risk in later life, with changes in both metabolism and blood pressure. In those born closer to, or at term, gestation was no longer relevant and an independent impact of preeclampsia on blood pressure was evident, so that by age 20, those born at term to preeclamptic pregnancies were four and a half times more likely to demonstrate clinically-apparent hypertension. I then investigated whether there were changes in other features of cardiovascular phenotype, independent of blood pressure, in preterm neonates born following preeclampsia (n = 46). At 3 months of age preterm infants born to hypertensive pregnancies had subclinical alterations in cardiac strain, independent of gestation or birth weight but not differences in blood pressure, or microvascular structure. These findings highlight preeclampsia and prematurity as key, independent perinatal factors, important in determining cardiovascular phenotype and risk during early life. Preeclampsia is associated with a specific lean, hypertensive phenotype, associated with cardiac functional alterations; these findings begin to define a distinct at risk population who may require targeted preventative interventions.

Thesis (PhD)-- Stellenbosch University, 2014. / ENGLISH ABSTRACT: Exposure to harmful stimuli can render vascular endothelial cells dysfunctional, characterised by reduced nitric oxide (NO) bioavailibility. Endothelial dysfunction (ED) is a reversible precursor of ischaemic heart disease (IHD), and understanding the mechanisms underlying the development of ED could lead to clinical strategies in preventing/treating IHD. Very little is known about the responses of cardiac microvascular endothelial cells (CMECs) to pro-ED stimuli, as most studies are conducted on macrovascular endothelial cells. The current dissertation set out to comprehensively investigate the responses of cultured primary adult rat CMECs to known harmful stimuli, viz. hypoxia and tumor necrosis factor-alpha (TNF-α; proinflammatory cytokine). We were interested to investigate whether this distinct endothelial cell type would develop classical features of ED, and if so, what the underlying mechanisms were. First we aimed to establish a baseline characterization of the CMECs under control conditions. Next, we developed a model of hypoxia-induced cell injury and measured apoptosis/necrosis, intracellular NO and reactive oxygen species (ROS), expression and activation of signalling proteins involved with NObiosynthesis, hypoxia and apoptosis, and differential regulation of proteins. Finally, we characterised CMEC responses to treatment with TNF-α. We assessed apoptosis/necrosis, intracellular NO and ROS levels, NO-biosynthesis pathway proteins and large-scale differential protein regulation. The above measurements were performed by morphological assessment (light and fluorescence microscopy), FACS analysis, western blotting and large-scale proteomic analyses. Data showed that CMECs shared many baseline features with other endothelial cell types, including morphological appearance, LDL-uptake, NO-production, and expression of eNOS protein. In a novel observation, proteomic analysis revealed the expression of 1387 proteins. Another novel finding was the high abundance of structural mitochondrial proteins, suggesting that CMECs require mitochondria for non-respiration purposes as well. High expression of vesicle, glycolytic and RAS signalling proteins were other features of the baseline CMECs. CMECs exposed to hypoxia responded by increased apoptosis/necrosis and expression of the hypoxia-marker, HIF-1α. Interestingly, hypoxic CMECs showed increased eNOS-NO biosynthesis, associated with increased mitochondrial ROS and reduced anti-oxidant systems, suggestive of oxidative stress. In accordance with the literature, several glycolytic proteins were up-regulated. A novel finding was the up-regulation of proteins involved with protein synthesis, not usually described in hypoxic cell studies. The CMECs responded to TNF-α-treatment by exhibiting hallmarks of ED, namely attenuated biosynthesis of PKB/Akt-eNOSderived NO and the development of outspoken response to oxidative stress as indicated by the up-regulation of several anti-oxidant systems. The data showed that TNF-α treatment elicited classical TNF-Receptor 1-mediated signalling characterized by the dual activation of pro-apoptotic pathways (BID and caspase-3) as well as the protective, pro-inflammatory IKB-alpha–NF-KB pathway. In conclusion, this is the first study of its kind to describe a comprehensive characterisation of CMECs under baseline and injury-inducing conditions. On the whole, although it appeared as if the CMECs shared many responses and mechanisms with more frequently researched endothelial cell types, the data also supplied several novel additions to the literature, particularly with the application of proteomics. We believe that this dissertation has provided more insights into endothelial heterogeneity in the vascular system and into the mechanisms adopted by CMECs when exposed to stimuli typically associated with cardiovascular risk. / AFRIKAANSE OPSOMMING: Blootstelling aan skadelike stimuli kan tot disfunksionaliteit van vaskulêre endoteelselle lei wat deur verlaagde biobeskikbaarheid van stikstofoksied (NO) gekenmerk word. Endoteeldisfunksie (ED) is ‘n omkeerbare voorganger van isgemiese hartsiekte (IHD), en ‘n beter begrip van die onderliggende meganismes van ED kan lei tot die ontwikkeling van kliniese strategieë vir die voorkoming/behandeling van IHD. Baie min is bekend oor die respons wat in kardiale mikrovaskulêre endoteelselle (CMECs) uitgelok word na blootstelling aan pro-ED stimuli, omdat meeste studies op makrovaskulêre endoteelselle uitgevoer word. Die huidige proefskrif het daarna gemik om die respons van primêre kulture van volwasse rot CMECs op bekende skadelike stimuli, nl. hipoksie en tumor nekrose faktor-alfa (TNF-α; pro-inflammatoriese sitokien) in diepte te ondersoek. Ons was veral geïnteresseerd om vas te stel of hierdie spesifieke endoteelseltipe die klassieke kenmerke van ED sou ontwikkel, en indien wel, wat die onderliggende meganismes sou wees. Eerstens het ons beoog om ‘n basislyn karaterisering van CMECs onder kontrole toestande daar te stel. Vervolgens het ons ‘n model van hipoksie-geïnduseerde selskade gevestig en apoptose/nekrose, intrasellulêre NO en reaktiewe suurstofspesies (ROS), sowel as die uitdrukking en aktivering van proteine betrokke by NO-biosintese, hipoksie en apoptose en differensiële regulering van proteine gemeet. Laastens het ons die respons van CMECs op behandeling met TNF-α gekarakteriseer. Ons het apoptose/nekrose, intrasellulêre NO en ROS vlakke, NO-biosintese-seintransduksieproteïene en grootskaalse differensiele regulering van proteïene gemeet. Bg. metings is uitgevoer deur gebruik te maak van morfologiese evaluasie (lig -en fluoressensiemikroskopie), vloeisitometriese analises, western blot analises en proteomiese analises. Data het getoon dat die basislyn eienskappe van CMECs grootliks met dié van ander endoteelseltipes ooreenstem, insluitende morfologiese voorkoms, LDL-opname, NO-produksie en die uitdrukking van eNOS proteïen. In ‘n nuwe waarneming, het die proteomiese data die uitdrukking van 1387 proteïene aangetoon. ‘n Ander nuwe bevinding was die voorkoms van ‘n groot aantal strukturele mitokondriale proteïene, wat daarop dui dat die CMECs mitokondria ook vir nie-respiratoriese doeleindes gebruik. ‘n Hoë uitdrukking van vesikulêre, glikolitiese en RAS-seintransduksie proteïene was ook kenmerkend van die basislyn CMECs. CMECS wat aan hipoksie blootgestel is, het reageer met ‘n verhoging in apoptose / nekrose en verhoogde uitdrukking van die hipoksie merker, HIF-1α. ‘n Interressante bevinding was dat eNOS-NO biosintese sterk toegeneem het in die hipoksiese CMECs wat met verhoogde mitokondriale ROS en verlaagde anti-oksidant sisteme (aanduidend van oksidatiewe stres) gepaardgegaan het. In ooreenstemming met die literatuur, is verskeie glikolitiese proteïene opgereguleer. ‘n Nuwe waarneming was die opregulering van proteïene wat betrokke is by proteïensintese, iets wat nie normaalweg in hipoksie-studies beskryf word nie. Die CMECs het op TNF-α behandeling gerespondeer deur tekens van ED te toon, naamlik ‘n afname in die NO afkomstig van PKB/Akt-eNOS biosintese en die ontwikkeling van uitgesproke reaksie op oksidatiewe stres soos aangedui deur die opregulering van verskeie anti-oksidant sisteme. Die data het ook aangedui dat TNF-α behandeling tot klassieke TNF-reseptor 1 bemiddelde seintransduksie gelei het, wat gekenmerk was deur die tweeledige aktivering van pro-apoptotiese seintransduksiepaaie (BID en kaspase-3) sowel as die beskermende, pro-inflammatoriese IKB-alpha-NF-KB seintransduksiepad. Ten slotte: hierdie is die eerste studie van sy soort wat die kenmerke en response van CMECs onder basislyn en pro-besering omstandighede in diepte beskryf. Alhoewel dit oor die algemeen wil voorkom asof die CMECs baie in gemeen het met ander, beter nagevorste endoteelseltipes, het die data egter ook verskeie nuwe bevindinge tot die bestaande literatuur gevoeg, spesifiek die data afkomstig van die proteomiese analises. Ons glo dat hierdie proefskrif meer insig verleen t.o.v. die heterogeniteit van vaskulêre endoteelselle asook t.o.v. die megansimes wat deur CMECs aangewend word wanneer hulle aan skadelike stimuli (geassosieer met kardiovaskulêre risiko) blootgestel word.

L’IRM cardiaque a une capacité unique d’étudier le remodelage ventriculaire gauche (VG) après infarctus du myocarde. Les objectifs principaux de ce travail étaient de caractériser le tissue de l’infarctus par IRM et d’évaluer les facteurs associés au remodelage du VG. Nous avons étudié prospectivement 114 patients présentant un premier infarctus du myocarde avec sus-décalage du segment ST et ayant subi une angioplastie primaire. Des IRM cardiaques ont été réalisées dans les 2 à 4 jours et à 6 mois suivant la revascularisation. Premièrement, nous avons réalisé une analyse comparative de différentes méthodes de segmentation de l’infarctus sur l’imagerie de rehaussement tardive (RT). Deuxièmement, nous avons étudié l’évolution des différentes composantes de la zone RT au cours des six mois, et observé que la réduction de la zone RT (33,8%) était représentée par l’extension de la zone grise initiale. Troisièmement, nous avons évalué le rôle clinique de la zone grise. Elle s’est révélée protectrice vis-à-vis du remodelage délétère. Quatrièmement, nous avons étudié l’influence de l’obstruction microvasculaire (OMV) sur le remodelage local du VG. Différents motifs ont été observés entre les patients atteints de l’OMV et ceux ne présentant pas d’OMV: un rétrécissement uniforme à travers le VG chez les patients sans OMV lorsque les sujets avec OMV présentaient une dilatation globale significative, ainsi qu’une dilatation plus importante dans les régions atteint d’OMV / Cardiac MRI (CMR) has the unique ability to study left ventricular remodeling after myocardial infarction. The main objectives of this work were to characterize infarct tissue by CMR and to evaluate factors associated with LV remodeling. We prospectively studied 114 patients with a first ST-segment elevation myocardial infarction (STEMI) undergoing primary angioplasty. CMR was performed within 2-4 days and at 6 months after the revascularization. First, we compared different methods for the segmentation of myocardial infarcts on late gadolinium enhancement (LGE) imaging. Second, we described the evolution of different components of LGE area during 6 months. We found that the decrease of LGE area (33.8%) matched the extent of initial gray zone. Third, we studied the clinical role of gray zone. The gray zone was found to be a protective factor for adverse remodeling. Fourth, we studied the influence of microvascular obstruction (MVO) on local LV remodeling and observed distinct remodeling patterns in patients with and without MVO: equally-distributed shrinkage throughout the LV cavity in patients without MVO whereas significant dilation occurring in those with MVO, tending to be greater in myocardial regions containing MVO

Depuis le milieu du XXème siècle, les techniques d’imagerie fonctionnelles ont un rôle grandissant dans notre compréhension sur les fonctions du cerveau en conditions physiologique et pathologique. Bien que l’IRMf fasse partie des techniques les plus communément utilisées pour l’imagerie du cerveau complet lors d’études préclinique et clinique, cette modalité souffre de sa résolution spatiotemporelle et sa sensibilité pour enregistrer finement les fonctions et activités cérébrales. Récemment l’imagerie fonctionnelle par ultrason (ifUS) a subi des développements permettant d’être complémentaires à l’IRMf ainsi qu’aux autres techniques d’imagerie cérébrales classiquement employées. Contrairement aux ultrasons focalisés conventionnels, l’imagerie hémodynamique proposé par l’ifUS repose sur une illumination ultrasonore plane permettant la détection des globules rouges en mouvement et la mesure de leur vitesse dans les micro-vaisseaux cérébraux. De ce fait, l’ifUS est indirectement lié à l’activité cérébrale d’où l’importance d’une meilleure compréhension des mécanismes du couplage neuro-vasculaire liant l’activité neuronale et les variations cérébrales d’apport en sang. De plus, cette technique a le potentiel pour fournir des informations précises sur les processus de certaines pathologies à la fois sur des modèles précliniques et chez l’homme. Dans un premier temps, j’exposerais mes travaux sur les récents développements techniques permettant l’ifUS in vivo (i) en condition chronique, (ii) sur l’animal éveillé, libre de mouvement et effectuant une tache comportementale et (iii) des vaisseaux capillaires chez le rongeur et l’homme. Dans un second temps, je démontrerais que l’ifUS in vivo peut fournir des informations nouvelles sur des pathologies telles que l’accident vasculaire cérébrale. / Since the middle of the 20th century, functional imaging technologies are making an increasing impact on our understanding on brain functions in both physiological and pathological conditions. Even if fMRI is nowadays one of the most used tool for whole brain imaging in pre-clinical and clinical studies, it lacks sufficient spatiotemporal resolution and sensitivity to assess fine brain function and activity. Functional ultrasound imaging (fUSi) has been recently developed and presents a potential to complement fMRI and other existing brain imaging modalities. Contrary to conventional ultrasound using focus beams, fUSi relies on hemodynamic imaging based on ultrasound plane-wave illumination to detect red blood cells movement and velocity in brain micro-vessels. Consequently, the fUSi signal is indirectly related to brain activity and it is therefore important to better understand the mechanisms of the neurovascular coupling linking neural activity and cerebral blood changes. Here again, fUSi may provide relevant information about disease processes in preclinical models but also in humans. First, I will present recent technical developments allowing in vivo fUSi (i) in chronic condition, (ii) in freely moving and behaving rats and (iii) in rodents and human brain capillaries. Second, I will demonstrate how fUSi could provide new insights in brain pathologies such as stroke.

Cardiovascular diseases (CVD) are the leading cause of death, and the underlying atherosclerotic process has its origin in youth. Physical activity lowers future CVD risk, however few adolescents achieve the recommended minimum amount of daily activity and interventions fail to meaningfully increase activity levels in this group. It is therefore essential to identify how small volumes of exercise can be optimised for the primary prevention of CVD. The purpose of this thesis is to identify the influence of exercise intensity on vascular health outcomes in adolescents, and to assess the efficacy of 2 weeks of low volume, high-intensity interval training on CVD risk factors in this population. Chapter 4 demonstrates that a single bout of high-intensity interval exercise (HIIE) performed one hour before a high fat meal elicits comparable reductions in postprandial lipaemia as a work-matched bout of moderate-intensity exercise (MIE) in girls. However, neither exercise attenuated postprandial lipaemia in the boys. Additionally, HIIE elicited a superior increase in postprandial fat oxidation and decrease in blood pressure, and this was sex independent. These findings are furthered in Chapter 5, which identified that accumulating HIIE, but not MIE, favourably modulates glycaemic control, postprandial blood pressure and fat oxidation in adolescents irrespective of sex. A high fat meal was included in Chapter 6 in order to impair vascular function via oxidative stress. Postprandial vascular function was preserved following MIE, but improved after HIIE, and these changes were not related to changes in postprandial lipaemia or total antioxidant status. Chapter 7 addressed the time course of the changes in vascular function post exercise, and identified that HIIE promotes superior changes in vascular function than MIE. Finally, Chapter 8 identified that 2 weeks of high-intensity interval training improved novel (endothelial function and heart rate variability), but not traditional CVD factors in adolescent boys and girls. However, most of these favourable changes were lost 3 days after training cessation. Thus, this thesis demonstrates that vascular health outcomes are positively associated with exercise intensity. Given that HIIE was perceived to be more enjoyable than MIE in Chapters 4, 6 and 7, performing HIIE appears to be an effectual and feasible alternative to MIE for the primary prevention of CVD.

We assessed potential mechanisms that may jeopardize skeletal muscle perfusion during surgery leading to adverse outcomes including muscle injury and flap hypoxia. In craniotomy patients, we observed an increase in serum lactate and creatine kinase and urine myoglobin; indicative of muscle damage. The early rise in lactate correlated with elevated BMI, suggesting that obesity caused tissue compression and muscle ischemia. In our rodent model, we investigated the effects of flap preparation and phenylephrine on muscle perfusion by assessing microvascular blood flow and tissue PO2. Phenylephrine reduced muscle blood flow by ~20%, yet increased PO2 by ~10% suggestive of decreased O2 metabolism. At baseline, muscle flap blood flow was reduced by ~50% while PO2 was severely reduced ~80% (~5 torr) suggesting that flap perfusion was attenuated and O2 metabolism was increased. Phenylephrine infusion further reduced muscle flap perfusion. These data demonstrate multiple mechanisms by which muscle perfusion is jeopardized during surgery.