The central role of the neutrophil in ischaemic/reperfusion injury

Inadequate blood flow to an organ, ischaemia, may lead to both local and remote tissue injury characterized by oedema, increased microvascular permeability to protein and degradation of connective tissue components. This damage is probably caused by the accumulation and inappropriate activation of neutrophils which occurs when the tissue is reperfused. To test this hypothesis a number of in vitro models of the sequential stages of ischaemia/reperfusion injury were examined. Methods were initially developed to examine the adhesion of neutrophils to monolayers of a cultured endothelial cell line (ECV304) after periods of hypoxia and reoxygenation. Neutrophil migration in response to factors secreted by the treated endothelial cells was then assessed. The genesis of an inappropriate oxidative burst by the neutrophil upon exposure to endothelial chemoattractants and adhesion molecules was also measured. Finally to appraise how tissue function might be affected by endothelial cell hypoxia the contractility of vascular smooth muscle was examined. Neutrophil adhesion to ECV304 cells, which had been hypoxic for 4 hours and then reoxygenated for 30 minutes, was significantly increased. This response was probably initiated by reactive oxygen species (ROS) generated by the endothelial cells. Blockage of their production by allopurinol reduced the heightened adhesion. Similarly removal of ROS by superoxide dismutase or catalase also attenuated adhesion. ROS generation in turn caused the release of a soluble factor (s) which induced a conformational change on the neutrophil surface allowing it to bind to the intercellular adhesion molecule 1 (ICAM-1) on the endothelial cell. Soluble factor (s) from hypoxia/reoxygenated endothelial cells also had a powerful neutrophil chemoattractant ability. When neutrophils were exposed to both hypoxic/reoxygenated endothelial cells and the soluble factor (s) released by them a large oxidative burst was elicited. This response was greatest immediately after reoxygenation and one hour later was diminishing suggesting at least one of the components involved was labile. Analysis of the supernatant from hypoxic/reoxygenated endothelial cell cultures and studies using inhibitors of secretion suggested platelet activating factor (PAF) may be a major component in this overall sequence of events. Lesser roles for IL-8, TNF and LTB4 were also suggested. The secretory products from hypoxia/reoxygenated endothelial cells also affected smooth muscle contractility having an anti-vasoconstrictor or relaxation property, similar to that exerted by PAF.

Glutaredoxin-1 up-regulation induces soluble vascular endothelial growth factor receptor 1, attenuating post-ischemia limb revascularization

Glutaredoxin-1 (Glrx) is a cytosolic enzyme that regulates diverse cellular function by removal of GSH adducts from S-glutathionylated proteins including signaling molecules and transcription factors. Glrx is up-regulated during inflammation and diabetes. Glrx overexpression inhibits VEGF-induced endothelial cell (EC) migration. The aim was to investigate the role of up-regulated Glrx in EC angiogenic capacities and in vivo revascularization in the setting of hind limb ischemia. Glrx overexpressing EC from Glrx transgenic mice (TG) showed impaired migration and network formation and secreted higher level of soluble VEGF receptor 1 (sFlt), an antagonizing factor to VEGF. After hind limb ischemia surgery Glrx TG mice demonstrated impaired blood flow recovery, associated with lower capillary density and poorer limb motor function compared to wild type littermates. There were also higher levels of anti-angiogenic sFlt expression in the muscle and plasma of Glrx TG mice after surgery. Non-canonical Wnt5a is known to induce sFlt. Wnt5a was highly expressed in ischemic muscles and EC from Glrx TG mice, and exogenous Wnt5a induced sFlt expression and inhibited network formation in human microvascular EC. Adenoviral Glrx-induced sFlt in EC was inhibited by a competitive Wnt5a inhibitor. Furthermore, Glrx overexpression removed GSH adducts on p65 in ischemic muscle and EC, and enhanced nuclear factor kappa B (NF-kB) activity which was responsible for Wnt5a-sFlt induction. Taken together, up-regulated Glrx induces sFlt in EC via NF-kB -dependent Wnt5a, resulting in attenuated revascularization in hind limb ischemia. The Glrx-induced sFlt may be a part of mechanism of redox regulated VEGF signaling.

Theoretical Investigation of Intra- and Inter-cellular Spatiotemporal Calcium Patterns in Microcirculation

Microcirculatory vessels are lined by endothelial cells (ECs) which are surrounded by a single or multiple layer of smooth muscle cells (SMCs). Spontaneous and agonist induced spatiotemporal calcium (Ca2+) events are generated in ECs and SMCs, and regulated by complex bi-directional signaling between the two layers which ultimately determines the vessel tone. The contractile state of microcirculatory vessels is an important factor in the determination of vascular resistance, blood flow and blood pressure. This dissertation presents theoretical insights into some of the important and currently unresolved phenomena in microvascular tone regulation. Compartmental and continuum models of isolated EC and SMC, coupled EC-SMC and a multi-cellular vessel segment with deterministic and stochastic descriptions of the cellular components were developed, and the intra- and inter-cellular spatiotemporal Ca2+ mobilization was examined. Coupled EC-SMC model simulations captured the experimentally observed localized subcellular EC Ca2+ events arising from the opening of EC transient receptor vanilloid 4 (TRPV4) channels and inositol triphosphate receptors (IP3Rs). These localized EC Ca2+ events result in endothelium-derived hyperpolarization (EDH) and Nitric Oxide (NO) production which transmit to the adjacent SMCs to ultimately result in vasodilation. The model examined the effect of heterogeneous distribution of cellular components and channel gating kinetics in determination of the amplitude and spread of the Ca2+ events. The simulations suggested the necessity of co-localization of certain cellular components for modulation of EDH and NO responses. Isolated EC and SMC models captured intracellular Ca2+ wave like activity and predicted the necessity of non-uniform distribution of cellular components for the generation of Ca2+ waves. The simulations also suggested the role of membrane potential dynamics in regulating Ca2+ wave velocity. The multi-cellular vessel segment model examined the underlying mechanisms for the intercellular synchronization of spontaneous oscillatory Ca2+ waves in individual SMC. From local subcellular events to integrated macro-scale behavior at the vessel level, the developed multi-scale models captured basic features of vascular Ca2+ signaling and provide insights for their physiological relevance. The models provide a theoretical framework for assisting investigations on the regulation of vascular tone in health and disease.

STRUCTURAL AND FUNCTIONAL CORRELATES OF EXERCISE VENTILATORY INEFFICIENCY IN CHRONIC OBSTRUCTIVE PULMONARY DISEASE PATIENTS WITH MILD-TO-MODERATE SPIROMETRIC ABNORMALITIES

Background: Individuals with chronic obstructive pulmonary disease (COPD) have higher than normal ventilatory equivalents for carbon dioxide (VE/VCO2) during exercise. There is growing evidence that emphysema on thoracic computed tomography (CT) scans is associated with poor exercise capacity in COPD patients with only mild-to-moderate airflow obstruction. We hypothesized that emphysema is an underlying cause of microvascular dysfunction and ventilatory inefficiency, which in turn contributes to reduced exercise capacity. We expected ventilatory inefficiency to be associated with a) the extent of emphysema; b) lower diffusing capacity for carbon monoxide; c) a reduced pulmonary blood flow response to exercise; and d) reduced exercise capacity. Methods: In a cross-sectional study, 19 subjects with mild-to-moderate COPD (mean ± SD FEV1= 82 ± 13% predicted, 12 GOLD grade 1) and 26 age-, sex-, and activity-matched controls underwent a ramp-incremental symptom-limited exercise test on a cycle ergometer. Ventilatory inefficiency was assessed by the minimum VE/VCO2 value (nadir). A subset of subjects also completed repeated constant work rate exercise bouts with non-invasive measurements of pulmonary blood flow. Emphysema was quantified as the percentage of attenuation areas below -950 Housefield Units on CT scans. An electronic scoresheet was used to keep track of emphysema sub-types. Results: COPD subjects typically had centrilobular emphysema (76.8 ± 10.1% of total emphysema) in the upper lobes (upper/lower lobe ratio= 0.82 ± 0.04). They had lower peak oxygen uptake (VO2), higher VE/VCO2 nadir and greater dyspnea scores than controls (p<0.05). Lower peak O2 and worse dyspnea were found in COPD subjects with VE/VCO2 nadirs ≥ 30. COPD subjects had blunted increases in pulmonary blood flow from rest to iso-VO2 exercise (p<0.05). Higher VE/VCO2 nadir in COPD subjects correlated with emphysema severity (r= 0.63), which in turn correlated with reduced lung diffusing capacity (r= -0.72) and blunted changes in pulmonary blood flow from rest to exercise (r= -0.69) (p<0.01). Conclusions: Ventilation “wasted” in emphysematous areas is associated with reduced exercise ventilatory efficiency in mild-to-moderate COPD. Exercise ventilatory inefficiency links structure (emphysema) and function (gas transfer) to a key clinical outcome (reduced exercise capacity) in COPD patients with modest spirometric abnormalities.

Thirty minutes of handgrip exercise enhances brachial artery flow-mediated dilation in response to two different shear stress profiles.

The walls of blood vessels are lined with a single-cell layer of endothelial cells. As blood flows through the arteries, a frictional force known as shear stress is sensed by mechanosensitive structures on the endothelium. Short and long term changes in shear stress can have a significant influence on the regulation of endothelial function. Acutely, shear stress triggers a pathway that culminates in the release of vasodilatory molecules from the endothelium and subsequent vasodilation of the artery. This endothelial response is known as flow mediated dilation (FMD). FMD is used as an index of endothelial function and is commonly assessed using reactive hyperemia (RH)-FMD, a method which elicits a large, short lived increase in shear stress following the release of a brief (5 min) forearm occlusion. A recent study found that a short term exposure (30 min) to a sustained elevation in shear stress potentiates subsequent RH-FMD. FMD can also result from a more prolonged, sustained increase in shear stress elicited by handgrip exercise (HGEX-FMD). There is evidence to suggest that interventions and conditions impact FMD resulting from sustained and transient shear stress stimuli differently, indicating that HGEX-FMD and RH-FMD provide different information about endothelial function. It is unknown whether HGEX-FMD is improved by short term exposure to shear stress. Understanding how exercise induced FMD is regulated is important because it contributes to blood flow responses during exercise. The study purpose was therefore to assess the impact of a handgrip exercise (intervention) induced sustained elevation in shear stress on subsequent brachial artery (BA) HGEX-FMD. Twenty healthy male participants (22±3yrs) preformed a 30-minute HGEX intervention on two experimental days. BA-FMD was assessed using either an RH or HGEX shear stress stimulus at 3 time points: pre-intervention, 10 min post and 60 min post. FMD and shear stress magnitude were determined via ultrasound. Both HGEX and RH-FMD increased significantly from pre-intervention to 10 min-post (p<0.01). These findings indicate that FMD stimulated by exercise induced increases in shear stress is potentiated by short term shear stress exposure. These findings advance our understanding regarding the regulation of endothelial function by shear stress.

Does estrogen fluctuation throughout the menstrual cycle impact flow-mediated dilation during exercise in healthy premenopausal women?

The endothelium is the inner most layer of cells that lines all arteries. A primary function of endothelial cells is to regulate responses to increased blood flow and the resulting frictional forces or shear stress by producing factors such as nitric oxide that mediate arterial dilation (flow mediated dilation (FMD)). Menstrual cycle variations in estrogen (E2) have been shown to influence brachial artery (BA) FMD in response to transient increases in shear stress brought about by the release of a brief forearm occlusion (reactive hyperemia (RH)). FMD can also be assessed in response to a sustained shear stress stimulus such as that created with handgrip exercise (HGEX), and studies have shown that RH- and HGEX stimulated FMD provide unique information regarding endothelial function. However, the impact of menstrual phase on HGEX-FMD is unknown. Therefore, the purpose of this study was to determine the impact of cyclical changes in E2 levels on HGEX-FMD over two discrete phases of the menstrual cycle. FMD was assessed via ultrasound. 12 subjects (21 ± 2yrs) completed two experimental visits: (1) low estrogen phase (early follicular) and (2) High estrogen phase (late follicular). In each visit both RH- and HGEX-FMD (6 min handgrip exercise) were assessed. Results are mean ± SD. E2 increased from the low to the high estrogen phase of the menstrual cycle (low: 34 ± 8, high: 161 ± 113pg/mL, p = 0.004). There was no change in mean FMD between phases (RH-FMD: 7.7 ± 4.3% vs. 6.4 ± 3.1%, p = 0.139; HGEX-FMD: 4.8 ± 2.8% vs. 4.8 ± 2.3%, p = 0.979). The observation that both RH- and HGEX-FMD did not differ between phases indicates that menstrual cycle fluctuations in estrogen may not universally impact endothelial function in young, healthy premenopausal women. Further research is needed to improve our understanding of the mechanisms that underlie variability in the impact of menstrual phase on both transient and sustained FMD responses.

Amphetamine increases blood pressure and heart rate but has no effect on motor recovery or cerebral haemodynamics in ischaemic stroke: a randomized controlled trial (ISRCTN 36285333)

Amphetamine enhances recovery after experimental ischaemia and has shown promise in small clinical trials when combined with motor or sensory stimulation. Amphetamine, a sympathomimetic, might have haemodynamic effects in stroke patients, although limited data have been published. Subjects were recruited 3-30 days post ischaemic stroke into a phase II randomised (1:1), double blind, placebo-controlled trial. Subjects received dexamphetamine (5mg initially, then 10mg for 10 subsequent doses with 3 or 4 day separations) or placebo in addition to inpatient physiotherapy. Recovery was assessed by motor scales (Fugl-Meyer, FM), and functional scales (Barthel index, BI and modified Rankin score, mRS). Peripheral blood pressure (BP), central haemodynamics and middle cerebral artery blood flow velocity were assessed before, and 90 minutes after, the first 2 doses. 33 subjects were recruited, age 33-88 (mean 71) years, males 52%, 4-30 (median 15) days post stroke to inclusion. 16 patients were randomised to placebo and 17 amphetamine. Amphetamine did not improve motor function at 90 days; mean (standard deviation) FM 37.6 (27.6) vs. control 35.2 (27.8) (p=0.81). Functional outcome (BI, mRS) did not differ between treatment groups. Peripheral and central systolic BP, and heart rate, were 11.2 mmHg (p=0.03), 9.5 mmHg (p=0.04) and 7 beats/minute (p=0.02) higher respectively with amphetamine, compared with control. A non-significant reduction in myocardial perfusion (Buckberg Index) was seen with amphetamine. Other cardiac and cerebral haemodynamics were unaffected. Amphetamine did not improve motor impairment or function after ischaemic stroke but did significantly increase BP and heart rate without altering cerebral haemodynamics.

Red blood cell rheology in sepsis

Changes in red blood cell (RBC) function can contribute to alterations in microcirculatory blood flow and cellular dysoxia in sepsis. Decreases in RBC and neutrophil deformability impair the passage of these cells through the microcirculation. While the role of leukocytes has been the focus of many studies in sepsis, the role of erythrocyte rheological alterations in this syndrome has only recently been investigated. RBC rheology can be influenced by many factors, including alterations in intracellular calcium and adenosine triphosphate (ATP) concentrations, the effects of nitric oxide, a decrease in some RBC membrane components such as sialic acid, and an increase in others such as 2,3 diphosphoglycerate. Other factors include interactions with white blood cells and their products (reactive oxygen species), or the effects of temperature variations. Understanding the mechanisms of altered RBC rheology in sepsis, and the effects on blood flow and oxygen transport, may lead to improved patient management and reductions in morbidity and mortality.

Theoretical investigation of intra- and inter-cellular spatiotemporal calcium patterns in microcirculation

Microcirculatory vessels are lined by endothelial cells (ECs) which are surrounded by a single or multiple layer of smooth muscle cells (SMCs). Spontaneous and agonist induced spatiotemporal calcium (Ca2+) events are generated in ECs and SMCs, and regulated by complex bi-directional signaling between the two layers which ultimately determines the vessel tone. The contractile state of microcirculatory vessels is an important factor in the determination of vascular resistance, blood flow and blood pressure. This dissertation presents theoretical insights into some of the important and currently unresolved phenomena in microvascular tone regulation. Compartmental and continuum models of isolated EC and SMC, coupled EC-SMC and a multi-cellular vessel segment with deterministic and stochastic descriptions of the cellular components were developed, and the intra- and inter-cellular spatiotemporal Ca2+ mobilization was examined.^ Coupled EC-SMC model simulations captured the experimentally observed localized subcellular EC Ca2+ events arising from the opening of EC transient receptor vanilloid 4 (TRPV4) channels and inositol triphosphate receptors (IP3Rs). These localized EC Ca2+ events result in endothelium-derived hyperpolarization (EDH) and Nitric Oxide (NO) production which transmit to the adjacent SMCs to ultimately result in vasodilation. The model examined the effect of heterogeneous distribution of cellular components and channel gating kinetics in determination of the amplitude and spread of the Ca2+ events. The simulations suggested the necessity of co-localization of certain cellular components for modulation of EDH and NO responses. Isolated EC and SMC models captured intracellular Ca2+ wave like activity and predicted the necessity of non-uniform distribution of cellular components for the generation of Ca2+ waves. The simulations also suggested the role of membrane potential dynamics in regulating Ca2+ wave velocity. The multi-cellular vessel segment model examined the underlying mechanisms for the intercellular synchronization of spontaneous oscillatory Ca2+ waves in individual SMC. ^ From local subcellular events to integrated macro-scale behavior at the vessel level, the developed multi-scale models captured basic features of vascular Ca2+ signaling and provide insights for their physiological relevance. The models provide a theoretical framework for assisting investigations on the regulation of vascular tone in health and disease.^

Hypothermia protects brain mitochondrial function from hypoxemia in a murine model of sepsis

Sepsis is commonly associated with brain dysfunction, but the underlying mechanisms remain unclear, although mitochondrial dysfunction and microvascular abnormalities have been implicated. We therefore assessed whether cerebral mitochondrial dysfunction during systemic endotoxemia in mice increased mitochondrial sensitivity to a further bioenergetic insult (hyoxemia), and whether hypothermia could improve outcome. Mice (C57bl/6) were injected intraperitoneally with lipopolysaccharide (LPS) (5 mg/kg; n = 85) or saline (0.01 ml/g; n = 47). Six, 24 and 48 h later, we used confocal imaging in vivo to assess cerebral mitochondrial redox potential and cortical oxygenation in response to changes in inspired oxygen. The fraction of inspired oxygen (FiO2) at which the cortical redox potential changed was compared between groups. In a subset of animals, spontaneous hypothermia was maintained or controlled hypothermia induced during imaging. Decreasing FiO2 resulted in a more reduced cerebral redox state around veins, but preserved oxidation around arteries. This pattern appeared at a higher FiO2 in LPS-injected animals, suggesting an increased sensitivity of cortical mitochondria to hypoxemia. This increased sensitivity was accompanied by a decrease in cortical oxygenation, but was attenuated by hypothermia. These results suggest that systemic endotoxemia influences cortical oxygenation and mitochondrial function, and that therapeutic hypothermia can be protective.

Investigating Angiotensin II in cardiac remodelling and the counter-regulatory actions of Angiotensin-(1-9)

Cardiovascular diseases (CVDs) including, hypertension, coronary heart disease and heart failure are the leading cause of death worldwide. Hypertension, a chronic increase in blood pressure above 140/90 mmHg, is the single main contributor to deaths due to heart disease and stroke. In the heart, hypertension results in adaptive cardiac remodelling, including LV hypertrophy to normalize wall stress and maintain cardiac contractile function. However, chronic increases in BP results in the development of hypertensive heart disease (HHD). HHD describes the maladaptive changes during cardiac remodelling which result in reduced systolic and diastolic function and eventually heart failure. This includes ventricular dilation due to eccentric hypertrophy, cardiac fibrosis which stiffens the ventricular wall and microvascular rarefaction resulting in a decrease in coronary blood flow albeit an increase in energy demand. Chronic activation of the renin-angiotensin-system (RAS) with its effector peptide angiotensin (Ang)II plays a key role in the development of hypertension and the maladaptive changes in HHD. Ang II acts via the angiotensin type 1 receptor (AT1R) to mediate most of its pathological actions during HHD, including stimulation of cardiomyocyte hypertrophy, activation of cardiac fibroblasts and increased collagen deposition. The counter-regulatory axis of the RAS which is centred on the ACE2/Ang-(1-7)/Mas axis has been demonstrated to counteract the pathological actions of Ang II in the heart and vasculature. Ang-(1-7) via the Mas receptor prevents Ang II-induced cardiac hypertrophy and fibrosis and improves cardiac contractile function in animal models of HHD. In contrast, less is known about Ang-(1-9) although evidence has demonstrated that Ang-(1-9) also antagonises Ang II and is anti-hypertrophic and anti-fibrotic in animal models of acute cardiac remodelling. However, so far it is not well documented whether Ang-(1-9) can reverse established cardiac dysfunction and remodelling and whether it is beneficial when administered chronically. Therefore, the main aim of this thesis was to assess the effects of chronic Ang-(1-9) administration on cardiac structure and function in a model of Ang II-induced cardiac remodelling. Furthermore, this thesis aimed to investigate novel pathways contributing to the pathological remodelling in response to Ang II. First, a mouse model of chronic Ang II infusion was established and characterised by comparing the structural and functional effects of the infusion of a low and high dose of Ang II after 6 weeks. Echocardiographic measurements demonstrated that low dose Ang II infusion resulted in a gradual decline in cardiac function while a high dose of Ang II induced acute cardiac contractile dysfunction. Both doses equally induced the development of cardiac hypertrophy and cardiac fibrosis characterised by an increase in the deposition of collagen I and collagen III. Moreover, increases in gene expression of fibrotic and hypertrophic markers could be detected following high dose Ang II infusion over 6 weeks. Following this characterisation, the high dose infusion model was used to assess the effects of Ang-(1-9) on cardiac structural and functional remodelling in established disease. Initially, it was evaluated whether Ang-(1-9) can reverse Ang II-induced cardiac disease by administering Ang-(1-9) for 2-4 weeks following an initial 2 week infusion of a high dose of Ang II to induce cardiac contractile dysfunction. The infusion of Ang-(1-9) for 2 weeks was associated with a significant improvement of LV fractional shortening compared to Ang II infusion. However, after 4 weeks fractional shortening declined to Ang II levels. Despite the transient improvement in cardiac contractile function, Ang-(1-9) did not modulate blood pressure, LV hypertrophy or cardiac fibrosis. To further investigate the direct cardiac effects of Ang-(1-9), cardiac contractile performance in response to Ang-(1-9) was evaluated in the isolated Langendorff-perfused rat heart. Perfusion of Ang-(1-9) in the paced and spontaneously beating rat heart mediated a positive inotropic effect characterised by an increase in LV developed pressure, cardiac contractility and relaxation. This was in contrast to Ang II and Ang-(1-7). Furthermore, the positive inotropic effect to Ang-(1-9) was blocked by the AT1R antagonist losartan and the protein kinase A inhibitor H89. Next, endothelial-to-mesenchymal transition (EndMT) as a novel pathway that may contribute to Ang II-induced cardiac remodelling was assessed in Ang II-infused mice in vivo and in human coronary artery endothelial cells (HCAEC) in vitro. Infusion of Ang II to mice for 2-6 weeks resulted in a significant decrease in myocardial capillary density and this was associated with the occurrence of dual labelling of endothelial cells for endothelial and mesenchymal markers. In vitro stimulation of HCAEC with TGFβ and Ang II revealed that Ang II exacerbated TGF-induced gene expression of mesenchymal markers. This was not correlated with any changes in SMAD2 or ERK1/2 phosphorylation with co-stimulation of TGFβ and Ang II. However, superoxide production was significantly increased in HCAEC stimulated with Ang II but not TGFβ. Finally, the role of Ang II in microvesicle (MV)-mediated cardiomyocyte hypertrophy was investigated. MVs purified from neonatal rat cardiac fibroblasts were found to contain detectable Ang II and this was increased by stimulation of fibroblasts with Ang II. Treatment of cardiomyocytes with MVs derived from Ang II-stimulated fibroblasts induced cardiomyocyte hypertrophy which could be blocked by the AT1R antagonist losartan and an inhibitor of MV synthesis and release brefeldin A. Furthermore, Ang II was found to be present in MVs isolated from serum and plasma of Ang II-infused mice and SHRSP and WKY rats. Overall, the findings of this thesis demonstrate for the first time that the actions of Ang-(1-9) in cardiac pathology are dependent on its time of administration and that Ang-(1-9) can reverse Ang II-induced cardiac contractile dysfunction by acting as a positive inotrope. Furthermore, this thesis demonstrates evidence for an involvement of EndMT and MV signalling as novel pathways contributing to Ang II-induced cardiac fibrosis and hypertrophy, respectively. These findings provide incentive to further investigate the therapeutic potential of Ang-(1-9) in the treatment of cardiac contractile dysfunction in heart disease, establish the importance of novel pathways in Ang II-mediated cardiac remodelling and evaluate the significance of the presence of Ang II in plasma-derived MVs.

Passive control of a bi-ventricular assist device : an experimental and numerical investigation

For the last two decades heart disease has been the highest single cause of death for the human population. With an alarming number of patients requiring heart transplant, and donations not able to satisfy the demand, treatment looks to mechanical alternatives. Rotary Ventricular Assist Devices, VADs, are miniature pumps which can be implanted alongside the heart to assist its pumping function. These constant flow devices are smaller, more efficient and promise a longer operational life than more traditional pulsatile VADs. The development of rotary VADs has focused on single pumps assisting the left ventricle only to supply blood for the body. In many patients however, failure of both ventricles demands that an additional pulsatile device be used to support the failing right ventricle. This condition renders them hospital bound while they wait for an unlikely heart donation. Reported attempts to use two rotary pumps to support both ventricles concurrently have warned of inherent haemodynamic instability. Poor balancing of the pumps’ flow rates quickly leads to vascular congestion increasing the risk of oedema and ventricular ‘suckdown’ occluding the inlet to the pump. This thesis introduces a novel Bi-Ventricular Assist Device (BiVAD) configuration where the pump outputs are passively balanced by vascular pressure. The BiVAD consists of two rotary pumps straddling the mechanical passive controller. Fluctuations in vascular pressure induce small deflections within both pumps adjusting their outputs allowing them to maintain arterial pressure. To optimise the passive controller’s interaction with the circulation, the controller’s dynamic response is optimised with a spring, mass, damper arrangement. This two part study presents a comprehensive assessment of the prototype’s ‘viability’ as a support device. Its ‘viability’ was considered based on its sensitivity to pathogenic haemodynamics and the ability of the passive response to maintain healthy circulation. The first part of the study is an experimental investigation where a prototype device was designed and built, and then tested in a pulsatile mock circulation loop. The BiVAD was subjected to a range of haemodynamic imbalances as well as a dynamic analysis to assess the functionality of the mechanical damper. The second part introduces the development of a numerical program to simulate human circulation supported by the passively controlled BiVAD. Both investigations showed that the prototype was able to mimic the native baroreceptor response. Simulating hypertension, poor flow balancing and subsequent ventricular failure during BiVAD support allowed the passive controller’s response to be assessed. Triggered by the resulting pressure imbalance, the controller responded by passively adjusting the VAD outputs in order to maintain healthy arterial pressures. This baroreceptor-like response demonstrated the inherent stability of the auto regulating BiVAD prototype. Simulating pulmonary hypertension in the more observable numerical model, however, revealed a serious issue with the passive response. The subsequent decrease in venous return into the left heart went unnoticed by the passive controller. Meanwhile the coupled nature of the passive response not only decreased RVAD output to reduce pulmonary arterial pressure, but it also increased LVAD output. Consequently, the LVAD increased fluid evacuation from the left ventricle, LV, and so actually accelerated the onset of LV collapse. It was concluded that despite the inherently stable baroreceptor-like response of the passive controller, its lack of sensitivity to venous return made it unviable in its present configuration. The study revealed a number of other important findings. Perhaps the most significant was that the reduced pulse experienced during constant flow support unbalanced the ratio of effective resistances of both vascular circuits. Even during steady rotary support therefore, the resulting ventricle volume imbalance increased the likelihood of suckdown. Additionally, mechanical damping of the passive controller’s response successfully filtered out pressure fluctuations from residual ventricular function. Finally, the importance of recognising inertial contributions to blood flow in the atria and ventricles in a numerical simulation were highlighted. This thesis documents the first attempt to create a fully auto regulated rotary cardiac assist device. Initial results encourage development of an inlet configuration sensitive to low flow such as collapsible inlet cannulae. Combining this with the existing baroreceptor-like response of the passive controller will render a highly stable passively controlled BiVAD configuration. The prototype controller’s passive interaction with the vasculature is a significant step towards a highly stable new generation of artificial heart.

Can venous occlusion plethysmography be used to measure high rates of arterial inflow?

To investigate whether venous occlusion plethysmography (VOP) may be used to measure high rates of arterial inflow associated with exercise, venous occlusions were performed at rest, and following dynamic handgrip exercise at 15, 30, 45, and 60 % of maximum voluntary contraction (MVC) in seven healthy males. The effect of including more than one cardiac cycle in the calculation of blood flow was assessed by comparing the cumulative blood flow over one, two, three, or four cardiac cycles. The inclusion of more than one cardiac cycle at 30 and 60 % MVC, and more than two cardiac cycles at 15 and 45 % MVC resulted in a lower blood flow compared to using only the first cardiac cycle (P < 0.05). Despite the small time interval over which arterial inflow was measured (~1 second), this did not affect the reproducibility of the technique. Reproducibility (coefficient of variation for arterial inflow over three trials) tended to be poorer at the higher workloads, although this was not significant (12.7 ± 6.6 %, 16.2 ± 7.3 %, and 22.9 ± 9.9 % for the 15, 30, and 45 % MVC workloads; P=0.102). There was also a tendency for greater reproducibility with the inclusion of more cardiac cycles at the highest workload, but this did not reach significance (P=0.070). In conclusion, when calculated over the first cardiac cycle only during venous occlusion, high rates of FBF can be measured using VOP, and this can be achieved without a significant decrease in the reproducibility of the measurement.

Plasma ATP concentration and venous oxygen content in the forearm during dynamic handgrip exercise

Background: It has been proposed that adenosine triphosphate (ATP) released from red blood cells (RBCs) may contribute to the tight coupling between blood flow and oxygen demand in contracting skeletal muscle. To determine whether ATP may contribute to the vasodilatory response to exercise in the forearm, we measured arterialised and venous plasma ATP concentration and venous oxygen content in 10 healthy young males at rest, and at 30 and 180 seconds during dynamic handgrip exercise at 45% of maximum voluntary contraction (MVC). Results: Venous plasma ATP concentration was elevated above rest after 30 seconds of exercise (P < 0.05), and remained at this higher level 180 seconds into exercise (P < 0.05 versus rest). The increase in ATP was mirrored by a decrease in venous oxygen content. While there was no significant relationship between ATP concentration and venous oxygen content at 30 seconds of exercise, they were moderately and inversely correlated at 180 seconds of exercise (r = -0.651, P = 0.021). Arterial ATP concentration remained unchanged throughout exercise, resulting in an increase in the venous-arterial ATP difference. Conclusions: Collectively these results indicate that ATP in the plasma originated from the muscle microcirculation, and are consistent with the notion that deoxygenation of the blood perfusing the muscle acts as a stimulus for ATP release. That ATP concentration was elevated just 30 seconds after the onset of exercise also suggests that ATP may be a contributing factor to the blood flow response in the transition from rest to steady state exercise.

Intraaortic balloon pump counterpulsation and cerebral autoregulation : an observational study

The use of Intra-aortic counterpulsation is a well established supportive therapy for patients in cardiac failure or after cardiac surgery. Blood pressure variations induced by counterpulsation are transmitted to the cerebral arteries, challenging cerebral autoregulatory mechanisms in order to maintain a stable cerebral blood flow. This study aims to assess the effects on cerebral autoregulation and variability of cerebral blood flow due to intra-aortic balloon pump and inflation ratio weaning.