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Skeletal muscle is spatially heterogeneous in muscle fibre type composition and microvascular supply. The capacity to quantify this heterogeneity in skeletal muscle is not routinely performed for it’s a laborious and time consuming technique. We have developed a high throughput data pipeline that utilises the simultaneous immunohistochemical labelling of muscle fibre type and microvascular supply, as an input for a semi-automated analysis software package that allows for the analysis of fine morphometric indices of fibre type composition and the interactions with microvascular supply. We have successfully shown that regional variation in fibre type composition impacts the functional characteristics of a muscle. After successful characterisation of regional heterogeneity in both structure and function we sought to establish their influence in physiological (adaptive) angiogenesis. Utilising animal angiogenic models we have shown that shear stress driven angiogenesis is principally a stochastic response that does not promote improved oxygen delivery when we analyse the spatial heterogeneity of the neovasculature. Conversely, skeletal muscle overload (abluminal stretch of microvasculature) increases the homogeneity of the oxygen supply area of the capillary bed, suggesting a tissue driven angiogenic response that is not evident in shear stress. Spinal cord injury induced rarefaction of the capillary bed attempts to maintain a homogeneous distribution of fibre size and capillary supply. The combination therapy of epidural stimulation and locomotor training can ameliorate the phenotypic change and rarefied capillary bed seen with spinal cord injury to that of intact levels. Endurance and resistance exercise have a largely similar global genomic response following a chronic training regime, which we are able to replicate in animal models of exercise through indirect electrical stimulation. The shear stress and muscle overload driven angiogenic response have distinctly different angiogenic pathways that contain no commonly expressed networks.

Vascular remodelling in arterial disease is characterised by vascular smooth muscle cell and adventitial fibroblast (AF) proliferation and migration due to mechanical and oxidative stresses within the vessel wall, leading to enhanced growth factor signalling. Transforming growth factor β-1 (TGF-β1) is a pleiotropic growth factor that mediates vascular remodelling through eliciting phenotypic changes in AF, which may result from the generation of reactive oxygen species (ROS). Nrf2 is a key transcription factor that co-ordinates expression of endogenous antioxidant defense genes such as heme oxygenase-1 (HO-1) and plays an important role in cellular defense against the deleterious effects of ROS. This thesis has investigated whether TGF-β1 modulates redox-related signalling pathways, gene expression, cell phenotype and motility in AF derived from human aortas. Results from this study have demonstrated that TGF-β1 treatment resulted in an increase in HO-1 protein expression in AF, an effect that was partially diminished in cells where Nrf2 had been knocked-down using siRNA. In addition, TGF-β1 also enhanced AF migration, measured using time-lapse video microscopy. In the presence of superoxide dismutase (SOD), migration of AF was significantly abrogated suggesting that TGF-β1-mediated AF migration may be mediated by the generation of superoxide. Measurement of ROS generation revealed an increase in AF treated with TGF-β1, an effect that was partially attenuated in the presence of a NAD(P)H oxidase inhibitor or SOD. The ability of the dietary isothiocyanate, sulforaphane (SFN) to activate the Nrf2 pathway in AF was also investigated. Results revealed that SFN treatment increased HO-1 protein expression which was diminished in cells where Nrf2 had been silenced using siRNA, suggesting that HO-1 induction was Nrf2-dependent. SFN also caused increased nuclear accumulation of Nrf2 and binding to the antioxidant response element (ARE), suggesting the involvement of Nrf2 in ARE-mediated antioxidant gene expression. The ability of SFN to activate endogenous antioxidant defenses may be a potential therapeutic strategy to counteract pathophysiological processes following vascular injury and in the pathogenesis of atherosclerosis. Findings from this study provide novel insights in to the mechanisms underlying the contribution of redox signaling in AF that lead to vascular diseases and for interventions to modulate TGF-β1-induced adventitial oxidative stress.

Oxidative stress is associated with a number of cardiovascular diseases such as atherosclerosis and pulmonary hypertension. Previously, our group has shown that prostaglandin–F2α and hypoxia enhance total tyrosine phosphorylation, activate Src and cause SrcFK-dependent constriction. Combining these observations with the ROS-sensitivity of Src, lead me to investigate whether interaction between SrcFK and ROS makes a central contribution to vascular smooth muscle function. Intra-pulmonary Arteries (IPA) were dissected from rat lungs, protein was extracted and phosphorylation of known targets of Rho-kinase (MYPT-1) and MLCK (MLC20) as well as Src auto-phosphorylation (Tyr416) was measured. Protein-protein interactions were examined by immune-precipitation/co-immuno-precipitation. Contraction was measured in IPA mounted on a wire myograph. Primary smooth muscle cell lines, generated from IPAs were used to measure ROS production using the luminescent dye L-012 or were transfected with GFP-tagged RhoA or ARHGEF1 to visualise protein translocation in live cells. In this study, I demonstrate that U46619 and LY83583 induce ROS production in PASMCs. I also demonstrate that U46619 and LY83583 contractile responses in IPA are sensitive to both antioxidants and SrcFK inhibition. U46619, hypoxia and LY83583 also enhance SrcFK autophosphorylation, ROCK activity and MLC20 phosphorylation which are blocked by SrcFK inhibition, antioxidants (U46619 and LY83583) and mitochondrial inhibitors (hypoxia). Finally, U46619 and LY83583 promote reversible translocation of eGFP-RhoA or eGFPARHGEF1 in a SrcFK dependent and ROS dependent manner. U46619 also enhanced coimmunoprecipitation of ARHGEF1 with SrcFK. Taken together these results suggest SrcFK’s play a key role as ROS sensitive intermediates which enhance contractile responses via interactions with ARHGEF1 and enhanced ROCK activity.

There are situations, during surgery, in the immediate post-operative period, and in neonatal and adult intensive care units, where monitoring the blood supply to a certain organ or region of the body could be of great value. Doppler ultrasound has great potential for monitoring blood flow velocity because it is a reliable, noninvasive method and produces results in real time. In addition, the development of Doppler technology associated with the recent progress of electronics and computerization has widened the range of information that can be extracted from the Doppler signal. Despite its great potential, there are difficulties which prevent the establishment of blood flow monitoring as a valuable clinical tool. Amongst these difficulties the one which appears to be the most important is the problem of fixing the transducer to the patient in order to prevent misalignment between ultrasound beam and vessel caused by patient movement. A Doppler tracking instrument which is able to adjust its beam direction automatically to ensure correct alignment between the beam and vessel would be valuable in overcoming this difficulty. The work described in this thesis led to the design, construction, and testing of hardware and software for such an instrument, which consists of a feedback controlled phased array transceiver, driving a one dimension array continuous wave Doppler transducer. With its operation it was possible to demonstrate the principle of Doppler tracking, which can open a wide area of monitoring to clinical ultrasound. From the construction point of view, this equipment was useful in identifying some guide lines to be followed in order to turn it into a device which can be used as a clinical tool.

Eosinophils play a pivotal role in the pathogenesis of asthma and allergic disease. The accumulation and persistence of eosinophils at sites of inflammation are mediated at least in part by the extended survival of eosinophils in response to circulating hematopoietins IL-3, IL-5 and GM-CSF. The apoptosis and subsequent clearance of eosinophils without histotoxic mediator release is thought to be crucial in the resolution of airway inflammation in asthma. This study characterised the morphological and biochemical events of human eosinophil apoptosis in vitro and investigated the mechanism by which IL-5 induces eosinophil survival. Peripheral blood eosinophils have a distinct expression profile of Bcl-2 homologues, critical regulators of apoptosis, with detectable expression of pro-apoptotic family homologues Bax, Bcl-xs, Bim, Bak, Bid, and Bad, and anti-apoptotic homologue Bcl-xL, with little or no detectable Bcl-2 expression. Stimulation with IL-5 induced modest upregulation of Bcl-2 mRNA and protein, with no significant modulation of the other Bcl-2 homologues examined. Caspases are the conserved executioners of the apoptosis. Eosinophils endogenously expressed 'initiator' caspase-8 and -9, and 'effector' caspase-3, -6, and -7. Spontaneous eosinophil apoptosis involved caspase-independent translocation of Bax to the mitochondria, resulting in perturbation of the mitochondrial membrane, cytochrome c release, and subsequent activation of caspase-3, -6, -7, -8, and -9. IL-5 inhibited constitutive eosinophil apoptosis at a site upstream of Bax translocation to the mitochondria, thereby preventing cytochrome c release and caspase activation. Eosinophils constitutively expressed the conformationally active form of Bax diffusely in the cytosol, but predominantly in the nucleus. Apoptosis induced by Fas receptor ligation involved detectable activation of caspase-3 and -8, and caspase-dependent Bax translocation to the mitochondria, supporting classification of eosinophils as a Type II cell in terms of apoptotic control. The data implicate Bax and mitochondria as pivotal regulators of eosinophil apoptosis in response to diverse stimuli.

Within the regulation of blood pressure, the renin-angiotensin system (RAS) plays an essential role with AngII causing vasoconstriction, which increases the blood pressure. AngII is formed after ACE ar cleaved by AngI. We hypothesize that the blunting of AngII production with concomitant exercise reduces improvements in metabolic fitness by removing an important stimulus for capillary growth in exercised muscles. The aim of this research was to expose the pathway by which AngII and ACE are implicated in exercise-induced capillary growth of human muscle. We show that highest increases in indices of metabolic strain are observed after high intense exercise bouts and that the increase in AngII is ACE genotype dependent. Furthermore, subjects with the ACE-II genotype had a significant higher capillary perfusion in the finger after exercise. Our findings of our ACE inhibition study support the view that an angiotensin-regulated mechanism affects the hypoxia-specific gene response in peripheral muscle to endurance exercise and support the notion that the muscle’s transcript regulation by ACE inhibition is related to muscle oxygenation during exercise. This indicates that there is a shift in the activation of the gene program from muscle fibres to the surrounding interstitium after ACE inhibition. In a training study we identified a correlation between increases in VO2max and the changes in capillary-to-fibre-ratio. We further observed that variability in the cardiovascular response, based on VO2max and heart rate at rest, was related to the ACE I/D genotype. Our findings that physiological improvements after exercise are dependent on ACE levels, both due to different ACE genotypes and to taking ACE inhibitors could have clinical repercussions. It may explain the individual variation in the response to exercise rehabilitation in aerobic power and oxygen uptake. These differences in exercise-induced improvements have rarely been valued in pharmacological studies of hypertension and exercise rehabilitation.

Two models of simulated ischaemia/reperfusion were established in the rat cardiac cell line H9c2 and in human umbilical vein endothelial cells (HUVECs). In both cell types, p38 was activated during ischaemia and reperfusion while JNK was only activated during reperfusion. In H9c2 cells, ERK was active during ischaemia and reperfusion while it was not responsive in HUVECs.;The expression of the pro-angiogenic vascular endothelial growth factors (VEGFs) during H9c2 ischaemia/reperfusion was examined. Ischaemia did not have any significant effect on VEGF expression but reperfusion stimulated a strong rise in VEGF-A mRNA levels. This was entirely due to post-transcriptional regulation as there was an increase in VEGF-A mRNA stability but not rise in promoter activity or transcription rate. The increase in VEGF-A mRNA stability was dependent upon activation of the JNK and ERK pathways.;Expression of the VEGF receptors Flt-1 and KDR was studied during ischaemia/reperfusion in HUVECs. While the mRNA levels of both genes remained constant throughout, KDR protein expression was elevated during ischaemia and this was dependent on p38 activation.;Further genes responsive to ischaemia/reperfusion were identified by microarray. Genes found to be induced included the cytoprotective genes neuroglobin and diaphorase 4 and the angiogenesis inhibitor calreticulin. Repressed genes included the potential angiogenesis inhibitor thrombospondin 4.;This study shows that that ischaemia/reperfusion resulted in a rise in pro-angiogenic gene expression and that this is dependent on MAP kinase activity. This suggests that the MAP kinases may play a pivotal role in regulating angiogenesis during cardiac ischaemia/reperfusion.

Green tea catechins have been in use for some time as a curative for a number of disorders and illnesses, however their direct benefits are still disputed. In this thesis several computational techniques have been applied to systems involving green tea catechins in order to study their properties and better ascertain their supposed health benefits. Based on first principles data, classical force fields of four selected green tea catechins (-)-Epigallocatechin 3-Gallate (EGCg), (-)-Epicatechin 3- Gallate (ECg), (-)-Epigallocatechin-3-O-(3-O-Methyl)-Gallate (EGCmg) and (-)-Epigallocatechin (EGC) have been parametrised in order to examine their behaviour under different conditions. Classical Molecular Dynamics coupled with Metadynamics has been used to explore the potential energy surface of these molecules as a function of selected torsions deemed crucial to the conformation of the system. In depth analysis of their interactions under classical conditions has been carried out in gas phase, water and chloroform. The catechins EGCg and EGC were further examined using Density Functional Theory (DFT) to determine their radical properties which are closely linked to their antioxidant activity. By calculating their ionisation potentials, bond dissociation enthalpies and acidity in gas phase, water and benzene solution we find little difference in their energies, despite large structural differences, and we investigate the limit of DFT in accurately describing these systems. Classical simulations of the cardiac protein Troponin C (a protein known to be crucial to the process of heart contraction), in complex with EGCg, were carried out from the basis of previous experimental work and it was found that EGCg exerts a stabilising effect on the calcium ion mobility present in the system and may help regulate calcium signalling in the cardiac sarcomere. Using sparse experimental data as a guide, alternative proteinligand binding possibilities were uncovered and used to help validate and expand the current experimental knowledge on the troponin C - troponin I system, giving rise to two low affinity, non-specific binding sites for EGCg which also affect calcium mobility.

Protein phosphatase 2A (PP2A) holoenzymes are present in most cell types, including cardiac myocytes, where they provide a large proportion of serine (Ser, S) and threonine (Thr, T) phosphatase activity. PP2A family members comprise a catalytic C subunit, a scaffold A subunit and a B-type subunit, which regulates subcellular targeting, substrate specificity and catalytic activity. Previous studies in our laboratory showed that in adult rat ventricular myocytes (ARVM) the subcellular localization of the regulatory B56α subunit is altered in response to isoprenaline (ISO) stimulation. This encouraged further studies of B56 subunits in β-adrenergic regulation of cardiac PP2A, which form the basis of this PhD project. Immunoblot analysis of ARVM samples revealed that in this cell system regulatory B56α, - γ and -δ subunits are expressed at protein level. Protein expression of PP2A scaffold and catalytic subunits was also confirmed. The subcellular distribution and ISO-induced translocation of PP2A subunits was investigated by fractionation of ARVM. B56α was depleted from the myofilament/nuclear compartment and was enriched in the cytosolic/membrane compartment of ISO-stimulated myocytes. This was observed also for PP2A scaffold and catalytic subunits. Potential ISO-induced phosphorylation of B56δ, which is phosphorylated by protein kinase A (PKA) at S573 in non-cardiac cells, was explored. Use of the Phos-tagTM SDS-PAGE system suggested that B56δ was phosphorylated in ARVM in response to ISO stimulation. In further studies, by using a phospho-site-specific antibody, increased phosphorylation of S573 was revealed. Studies with propranolol, a non-selective β-adrenergic receptor (β-AR) antagonist, confirmed that the response is mediated by β-ARs. Studies with CGP 20712A (a β1-selective antagonist) and ICI 118,551 (a β2-selective antagonist) indicated that the response is mediated primarily by β1-ARs. Studies with compounds that inhibit (H89 and PKI) or activate (N6-Benz-cAMP) PKA showed that the activity of PKA is necessary and sufficient for the response. To explore the functional role of B56δ phosphorylation at S573, adenoviral vectors encoding GFP-tagged human B56δ in wild type (WT) or mutated form, in which S573 is replaced by non-phosphorylatable alanine (Ala, A), were constructed and post-infection protein expression profiles in ARVM were characterized. The studies confirmed that heterologous WT B56δ is phosphorylated at S573 in response to ISO stimulation and that heterologous S573A B56δ is not phosphorylated at this site. Overexpression of WT B56δ but not S573A B56δ appeared to amplify ISO-induced phosphorylation of some PKA substrates. Further studies are now necessary to identify the direct targets of B56δ-PP2A and the impact of altered B56δ phosphorylation, and in particular to determine the physiological role of B56δ phosphorylation at S573 in β-adrenergic regulation of cardiac function.