The effects of manganese (and some other transition elements) on the electrical and contractile properties of frog heart muscle have been investigated. The action potential duration and overshoot were dependent upon the intensity of stimulation in the presence of manganese. The cells were hyperpolarised by approximately 10 mV for a ten-fold increase in the manganese (or calcium concentration). This hyperpolarization could not completely account for the large increases in the threshold stimulus for contraction produced in manganese and high calcium solutions. The twitch contraction was rapidly and reversibly inhibited by manganese. The contracture responses produced by low-sodium solutions were greatly inhibited by manganese (I50 approximately 0.6 mM) and are largely accounted for by assuming a competitive inhibition of calcium binding by manganese. The drugs D-600 and verapamil produced little or no inhibition of the low-sodium contracture. Sodium, lithium, hydrazine and hydroxylamine were able to induce contraction in the presence of manganese following exposure to sodium-free conditions. Increasing the intracellular sodium concentration by exposure to low potassium solutions potentiated both the low-sodium contracture and the twitch contraction suggesting that intracellular sodium is important in the regulation of calcium influx. Manganese produced less inhibition of the potassium-rich contracture than of that produced by low-sodium solutions. The hyperpolarisation in manganese solutions is insufficient to account for the inhibition of the contractures produced by low-sodium or potassium-rich solutions. Contractures produced by the addition of caffeine were not inhibited by manganese. Measurements of the uptake of manganese by whole canulated ventricles indicated a large accumulation and a slow loss of part of the accumulated manganese on return to manganese-free solutions.

The murine retinal vasculature consists of three co-planar plexi, which develop in the first three weeks of postnatal life (PO-P20). The first to form is the superficial plexus: blood vessels migrate radially outwards from the optic nerve head (ONH) region and along the surface of the retina, guided by a template of astrocytes. This plexus reaches the retinal periphery at around P7, at which time, vertical vascular sprouts begin to project into the deep layers of the retina. Two further plexi then ramify around the inner and outer surfaces of the inner nuclear layer (INL), the deeper of which is the first to form (P7-PI2), whereas the intermediate plexus forms last (PI4-P20). During development, an immature, high calibre capillary-only plexus undergoes differentiation and remodelling to become a mature, low calibre, adult plexus with identifiable arteries and veins. In the first experimental chapter of this project, immunohistochemical (IHC) techniques were used to discover that arteriovenous differentiation is linked to a tightly-patterned process of capillary pruning by caspase-dependent endothelial cell apoptosis. IHC using EFs showed that specific spatio-temporal patterning of hypoxia occurs within the neuroblast layer during deep and intermediate plexus development. Muller glial cell (MGC) and R-Cadherin imaging showed that vertical sprouting of the plexus occurs under the guidance of MGC processes and that the spatio-temporal expression of R-Cadherin defines the locations of the plexi. The second experimental chapter showed that transgenic overexpression of vascular endothelial growth factor 188 (VEGF 188) in the eye ameliorates the vaso-obliteration and pathological neovascularisation associated with oxygen-induced retinopathy (OIR). In the third experimental chapter a novel transgenic mouse strain was created, which over-expresses the VEGFI6Sb isoform in the eye using the same promoter as the aforementioned transgenic mouse. Despite positive identification as transgenics, no ocular perturbations were found in this novel strain.

Human monocytes comprise three distinct subsets, defined by their relative expression of CD14 and CD16. These subsets appear to have different functional roles, which are only recently becoming delineated. Functional differences include the manner in which different monocytes interact with blood endothelial cells. Interaction with endothelial cells in the micro-vasculature controls access of monocytes to tissues. This interaction is of particular interest in inflammatory diseases such as Rheumatoid Arthritis (RA), in which inflammatory cell infiltration into target tissues causes the majority of the associated damage. The studies in this thesis aim to examine the molecular interactions occurring between monocytes of different subsets and the endothelium during different kinds of locomotion. A multi-step, systems-based approach was taken. First, analysis of macro- and micro-vascular endothelium by flow cytometry revealed differences in the cell surface expression of a range of well-described adhesion/function-associated molecules in both resting and activated states. Second, these differences were further investigated by gene array analysis of micro-vascular endothelium under resting and inflammatory conditions. In a limited number of clinical cases, endothelial cells were isolated from RA synovium and characterised by gene expression microarray. These data showed that similar cellular pathways are up-regulated by RA synovial endothelium and micro-vascular endothelium under in vitro inflammatory conditions. In the third step, micro-vascular endothelium gene expression was analyzed in parallel with that of the three major monocyte subsets in order to identify potential receptor-ligand binding pairs. Six putative receptor-ligand pairs were taken forward for functional analysis. In order to facilitate this, a novel in vitro migration assay was developed to enable simultaneous monitoring of the locomotory behaviour of all three monocyte types over endothelial monolayers by live-cell, fluorescent time-lapse imaging, with the whole system subjected to physiologically relevant shear flow. In this final fourth step, each monocyte subset was shown to preferentially perform different types of locomotory behaviour in a resting state. The roles of the six putative receptor-ligand pairs in each type of behaviour, for each monocyte subset, were then investigated using blocking reagents. As a result, the work in this thesis contributes to the functional delineation of human monocyte subsets and provides insight into the molecular basis for monocyte-endothelial interaction behaviour.

Background: Clinical studies have demonstrated platelet activation in inflammatory disorders including asthma and allergic rhinitis. This is distinct from platelet aggregation involved in the maintenance of haemostasis. Whilst signalling involved in platelet activation in haemostasis is well known, signalling pathways in activation responding to inflammatory stimuli remains unclear. Objectives: Here I investigated whether purinergic activation of platelets in allergic inflammation is distinct from purine involvement in platelet aggregation. In the second part, stimulation of platelets with a large range of both inflammatory and haemostatic stimuli was used to investigate potential distinct differences in platelet function. Methods: Balb/c mice were sensitised to Ovalbumin (OVA) and subsequent OVA challenge. Bronchoalveolar lavage fluid was analysed for inflammatory cells and blood samples were collected and analysed for platelet activation. The role of platelet purinergic receptors and associated signalling mechanisms (RhoA) were also assessed. Additional in vitro experiments were performed on isolated human platelets to investigate the impact of a range of inflammatory and haemostatic stimuli on platelet function through measurements on P-selectin expression, platelet-leukocyte conjugation, aggregation and migration. Results: Allergen challenge induced significant increases in pulmonary leukocyte recruitment compared to sham controls (P<0.001).P2Y1 (P < 0.001), but not P2Y12 or P2X1 antagonism inhibited allergen induced pulmonary leukocyte recruitment. The formation of platelet-leukocyte conjugates in vivo and platelet/P-selectin dependent polymorphonuclear cell migration in vitro was exclusively P2Y1 dependent, furthermore allergen challenge induced significant increases in RhoA activity, a process which was inhibited exclusively through P2Y1 receptor antagonism. Leukocyte recruitment remained significantly suppressed in thrombocytopenic mice following reinfusion of platelets treated with a P2Y1 antagonist or a Rho-associated kinase inhibitor confirming a crucial role for RhoA activity downstream of platelet P2Y1 receptors. Secondly, stimulation of platelets with chemotactic stimuli such as macrophage-derived chemokine (P < 0.01) and stromal-cell derived factor 1α (P < 0.001) induced significant platelet migration without increases in P-selectin, platelet-leukocyte conjugates or aggregation. Conversely haemostatic stimuli induced increases in all measured parameters bar platelet migration Conclusion: RhoA signalling downstream of platelet P2Y1, but not P2Y12, represents a clear dichotomy in platelet activation during allergic inflammation versus haemostasis. Furthermore, platelet activation by different stimuli (inflammatory or haemostatic) can induce differences in platelet function further emphasising a dichotomy in platelet function.

Background: Aging is a physiological process characterised by a progressive dysfunction in metabolism and impaired tissue repair capacity, eventually leading to organs’ and tissue degeneration. Low-grade inflammation is currently considered the main driver of aging. The measure of telomeres length in peripheral leukocytes (LTL) has been suggested as novel and reliable marker of aging. The aim of this PhD was to investigate the association between (LTL), chronic inflammation and common cardio-metabolic risk factors. Methods: Four studies were conducted: 1) a case-control analysis of 356 cases with periodontitis (PD) and 206 controls to assess the differences in LTL between cases and controls, 2) a cross sectional analysis of 630 individuals with diabetes mellitus investigating the association between PD, LTL and gluco-metabolic factors, 3) a cross-sectional analysis of 1080 adolescents (13–16 years old) to investigate the association between LTL, inflammation and cardiovascular (CV) disease risk factors and 4) a 10 years longitudinal analysis in 2547 women and 2815 men to assess whether LTL predicted cardiac and vascular phenotypes. LTL were measured using a Real Time Polymerase Chain Reaction method in all studies. Results: Study I demonstrated that increased systemic inflammation and oxidative stress were associated with shorter LTL in cases with PD versus controls (P<0.05). Results from Study II confirmed that shorter LTL was associated with severe periodontal inflammation (p=0.04), increased endotoxemia and insulin resistance. In Study III LTL was inversely associated with C-reactive protein (P<0.001) and fibrinogen (P=0.001) in adolescents. Lastly in Study IV a faster rate of telomere shortening between 53 to 60-64 years was associated with subclinical atherosclerosis at 60-64 years (p=0.006). All results were independent of traditional CV risk factors. Conclusions: This PhD programme provides evidence in support of the use of LTL as novel marker of aging and predictor of age-related diseases.

Natriuretic peptides are a family of vasoactive hormones that play important roles in cardiovascular homeostasis. These peptides exert biological effects primarily via activation of guanylate cyclase (GC)-coupled natriuretic peptide receptors (NPR) that generate the intracellular messenger cyclic guanosine 3',5'-monophosphate (cGMP). Activation of the cytosolic GC by NO is well-established to mediate cGMP-dependent, anti-atherogenic effects however, an analogous cytoprotective role for natriuretic peptides has yet to be fully elucidated. Since many cardiovascular disorders (e.g. atherosclerosis, septic shock) are now accepted as inflammation-based diseases, identification of potential roles for natriuretic peptides in regulating vascular inflammation might assist in the prevention and treatment of cardiovascular pathology. The studies described in this thesis investigated the hypothesis that natriuretic peptides (i.e. atrial natriuretic peptide ANP , C-type natriuretic peptide CNP ) affect pro-inflammatory protein expression (i.e. inducible NO synthase, iNOS) and cell proliferation via activation of GC-linked NPR. Herein, it is demonstrated that in NPR-A knockout mice, iNOS expression and NO production in response to intravenous administration of bacterial lipopolysaccharide is significantly reduced compared to wild-type controls this difference is mirrored in the ex vivo functional reactivity of vessels from such animals. However, neither ANP nor CNP were able to alter iNOS expression or NO production in vitro in RAW264.7 murine macrophages or primary rat aortic smooth muscle cells. CNP, but not ANP, transiently enhanced phosphorylation of extracellular signal-regulated kinase (ERK)1/2. CNP-induced ERK 1/2 phosphorylation was blocked by the selective ERK 1/2 inhibitor PD98059, the Gj-protein inhibitor Pertussis toxin, and the selective NPR-C antagonist M372049. Accordingly, CNP inhibited vascular smooth muscle proliferation in a PD98059- and M372049-reversible manner. These observations suggest that part of the anti-atherogenic profile of CNP is mediated via NPR-C, Gi-dependent ERK 1/2 phosphorylation and inhibition of vascular smooth muscle proliferation. Moreover, my findings identify a potential pro-inflammatory role for ANP/NPR-A-dependent signalling in vivo.

Congenital cardiovascular malformations (CCVM) are the most common human birth defects, occurring in up to 1% of newborn infants. Many cell populations are required for correct cardiovascular development, including the neural crest cells (NCC). NCCs migrate from the dorsal neural tube into the pharyngeal arches and contribute to the asymmetric remodelling of the pharyngeal arch arteries (PAA). A subset of NCCs continue to migrate into the outflow tract (OFT) of the heart, and aid in the septation of the OFT into the aorta and pulmonary trunk. Tcfap2a, which encodes the murine transcription factor AP-2α, is highly expressed within NCCs and the pharyngeal surface ectoderm (PSE), and mutations in this gene causes CCVM affecting the OFT, PAAs and the formation of the interventricular septum. This thesis investigates the tissue-specific requirements of Tcfap2a in cardiovascular development and the mechanisms of PAA malformation. Conditional deletion of Tcfap2a from NCCs resulted in a limited prevalence of CCVM, which was not increased by altering the genetic background or simultaneous deletion from the PSE. However, Tcfap2a expression within both the NCCs and PSE is required in craniofacial and thymus development. Immunohistochemical analysis in Tcfap2a-null embryos suggests that Tcfap2a is required in the formation of the PAAs, in addition to a possible role in their remodelling. Quantitative PCR analysis was used to investigate potential transcriptional targets of AP-2α within the pharyngeal arch region and we propose that Dlx5 is regulated by AP-2α in a pathway independent of endothelin signalling. A study investigating the potential interaction between these genes is presented here. This thesis presents further insights into the genetic networks regulating the formation of the PAAs, OFT and interventricular septum of the heart. We also highlight issues associated with the use of transgenic mouse models and the effect of genetic background in the study of cardiovascular development.

Platelet secretion not only drives thrombosis and haemostasis, but also mediates a variety of other physiological and pathological processes. The ubiquitous SNARE machinery and a number of accessory proteins have been implicated in regulating secretion in platelets. Although several platelet SNAREs have been identified, further members of the SNARE family may help fine-tune platelet secretion. In this study I identified expression of t-SNAREs VTIlA, VTIlB (Qb SNAREs) and STX8 (Qc SNARE) in human and mouse platelets. Those 'novel' SNAREs were able to interact with each other and previously reported SNAREs VAMP8 (R-SNARE) and STXll (Qa SNARE), thus suggesting existence of a secondary SNARE complex in addition to the widely accepted SNAP23-VAMP8- STXll complex. In following mouse studies, whereas neither gene was found to be essential for a -granule or lysosome secretion, Stx8-/- platelets showed a significant defect in dense granule secretion and aggregation, that was most pronounced at intermediate concentrations of agonists. Addition of exogenous ADP could rescue the aggregation defect but failed to restore dense granule secretion, suggesting the defect lies in the 'primary' secretory pathway. Pretreatment with P2Y receptors antagonists reduced secretion and aggregation to the same extent in WT and Stx8-/- platelets, suggesting that the ADP-driven positive feedback mechanism was not defective in Stx8-/- platelets. In addition, STX8 was found to play a role in regulating pro-coagulant activity suggesting novel roles for SNAREs in platelets. Neither Vtila-/- nor Vtilb-/- showed any significant defects, suggesting complementarity between those homologues in platelets. STX8 therefore specifically contributes to dense granule secretion and represents another member of a growing family of genes that play distinct roles in differentially regulating granule release from platelets. Taken together, data presented in this Thesis not only provides first evidence of an additional SNARE complex present in platelets but also suggests novel roles for SNAREs in regulation of thrombosis and haemostasis.

Platelet activation is essential in haemostasis and thrombosis and central to this role is cytosolic calcium (Ca2+) elevation. The two ways in which this can occur are release from intracellular Ca2+ stores and the entry of Ca2+ across the plasma membrane (PM). Changes in cytosolic Ca2+ are required to enable a variety of platelet responses. In platelets and other non-excitable cells, the major route of Ca2+ entry occurs as a result of Ca2+ depletion from intracellular stores, and is known as store operated Ca2+ entry (SOCE). STIM1 is the Ca2+ sensing protein of the endoplasmic reticulum (ER) which activates Orai channels in the plasma membrane allowing Ca + entry. However, not all of the details of Ca2+ entry are understood. In platelets, two other pathways also enable Ca2+ entry. The P2X1 receptor is a ligand gated ion channel activated by the binding of ATP. Second messengers (such as 1,2-diacyl-sn- glycerol [DAG]) and phosphatidylinositol-3,4,5-trisphosphate (PI-3,4,5-P3) may directly gate plasma membrane cation channels such as the transient receptor potential canonical 6 (TRPC6) channel. -- The main aim of this study was to examine the role of STIM1 and TRPC proteins in Ca2+ entry in human platelets. The TRPC1 protein was originally proposed to be a major component of the SOC channel based on the observation that a commercial preparation of an anti-TRPCl antibody against the channel impaired SOCE in platelets. However, in the current study I demonstrate the failure of this reagent to bind to TRPC1. The previously reported inhibitory effect on SOCE may be due in part to be a result of the azide component of the antibody preparation.

Central aortic systolic blood pressure (cSBP) differs from peripheral systolic blood pressure (pSBP) measured in the arm. cSBP may be estimated non-invasively by application of a generalised transfer function (GIF) to a high fidelity peripheral arterial waveform or from the late systolic shoulder (SBP2) of such a waveform. The relative accuracy of these estimates and the degree to which they depend on the accuracy of peripheral blood pressure is unknown. The interest in estimates of central blood pressure is driven in large part by the fact that aortic pulse pressure (cPP) is thought to be a better predictor of cardiovascular risk and response to antihypertensive treatment than peripheral BP. However, little is known concerning the mechanism by which drugs may reduce cPP independently of effects on peripheral BP. Objectives of this thesis were to: 1. Examine the relative accuracy of different methods (GTF and SBP2) for estimating cSBP and cPP from a high fidelity peripheral arterial waveform. 2. Determine errors introduced by non-invasive calibration of this waveform (as would be the case when such methods are used in practice), 3. Explore the use of a simplified method for estimating cSBP based upon pressure oscillations within an arm cuff. 4. Determine the mechanism by which nitroglycerin (NTG, a drug that has relatively selective actions to lower cSBP) lowers cPP. Pressure and in some cases combined pressure and flow velocity were acquired at the aortic root during cardiac catheterisation. Peripheral blood pressure was measured by oscillometry and peripheral blood pressure waveforms were obtained from blood pressure cuffs, radial tonometry and a servo-controlled finger cuff. To address objective 4 additional measurements of ventricular and arterial mechanics where made using ultrasound and magnetic resonance imaging.