Diffuse, multi vessel coronary artery disease (CAD) affects about one third of patients with CAD and is associated with worse outcomes. Abnormal vascular stiffness and function (e.g., reflected by increased endothelial microparticles and diminished microvascular endothelial-mediated responses), cell mediated pro-inflammatory status (e.g., reflected by levels of specific monocyte subsets), and platelet function (e.g., increased monocyte-platelet aggregates (MPAs) and platelet microparticles) have established roles in CAD pathogenesis but their contribution to the unfavourable diffuse CAD form is unclear. The aim of this study was to compare measures of vascular function, monocyte subsets, MP As, and endothelial and platelet microparticles in patients with diffuse and focal CAD and subjects without CAD. Additionally, prospective changes in these characteristics were analysed over one year. I found increased counts of aggregates of Mon2 monocyte subset with platelets and apoptotic endothelial microparticles in patients with diffuse CAD and I identified a negative correlation between Mon2 MPAs and microvascular endothelial function and increased diastolic elastance. My findings suggest that excessive levels of Mon2 aggregates with platelets and apoptotic endothelial micropa1iicles could be important contributors to the diffuse type of CAD by a mechanism involving microvascular endothelial dysfunction and abnonnal cardio-vascular interactions.

Recent studies have demonstrated a key role for platelets and microthrombi in the pathophysiology of intestinal ischaemia-reperfusion (IR) injury. The research described in this thesis investigates the role of the major platelet glycoprotein receptors in recruiting and activating platelets following intestinal IR injury, namely GPVI, GPIb-IX-V and the integrins IIb3 and 21. Intravital microscopy was utilised to monitor individual platelet adhesion and microthrombus formation in anaesthetised mice undergoing intestinal IR injury \(in\) \(vivo\) using a novel dual labelling methodology. This study focussed on the microcirculation of the mucosal villi as this luminal surface is most susceptible to IR injury. We demonstrate that it was necessary to inhibit both microthrombus formation as well as platelet-leukocyte-endothelial interactions in order to ensure longer lasting improvement in gut microcirculation and histology. This was achieved through a dual therapy that targeted both GPIb and P-selectin. The strongest anti-platelet effect was observed with blocking the IIb3 integrin, but this was also associated with a sustained bleeding from the mucosal surface. Overall, the research within this thesis suggests that therapeutic strategies targeting GPIb and P-selectin may prove beneficial in improving the clinical morbidity associated with gut IR injury.

The platelet receptor CLEC-2 is the only known endogenous ligand for the transmembrane receptor podoplanin, which is expressed on lymphatic endothelial cells (LEC) as well as a number of other cell types. Both CLEC-2 and podoplanin are required for normal lymphangiogenesis as mouse embryos lacking either protein develop a phenotype in which blood is detected in the lymphatic vessels. This thesis examines the role of the podoplanin-CLEC-2 interaction in the migratory and tube-forming capabilities of LEC. Addition of platelets or antibody-mediated podoplanin crosslinking both inhibited migration of LEC in transfilter migration assays in the presence, but not absence, of vascular endothelial growth factor (VEGF)-C. Similarly, platelets and podoplanin crosslinking reduced stability of LEC networks formed in co-cultures with fibroblasts. We also found that siRNA-mediated knockdown of podoplanin negated the pro-migratory effects of VEGF-C and VEGF-A. Furthermore, we obtained evidence that podoplanin signalling may involve RhoA and Rho-kinase, and that the effect of podoplanin might be linked to its phosphorylation by protein kinase A downstream of VEGF receptor signalling. These data suggest that the interaction of podoplanin and CLEC-2 prevents connection between blood and lymphatic vessels through reductions in LEC migration and stability of cell-cell interactions.

Numerous large clinical trials of cardiovascular risk lowering agents have been conducted in the hope of reducing the excess cardiovascular risk found in patients with diabetes mellitus. However, the relationship between glucose and cardiovascular disease remains complex and various areas require further study. Even in patients with diabetes, an individual’s cardiovascular risk is highly variable depending on other clinical characteristics, the assumption that glucose is a continuous risk factor has often been based on weak evidence from relatively short studies, the effect of commonly used cardiovascular risk lowering agents often has unexpected effects on new-onset diabetes and statins have not yet been studied in detail, and whether glucose-lowering therapies actually reduce cardiovascular risk has remained a contentious issue despite the conduct of large clinical trials. Furthermore, the realisation that the combination of diabetes and chronic heart failure, a common complication of coronary disease, carries a particularly poor prognosis suggests that prediction of diabetes in this population may be clinically valuable. Aims: I aimed to address the following different, though related, questions regarding glucose and cardiovascular disease: 1. Are anticipated cardiovascular event rates in diabetes endpoint trials actually achieved? Is it possible to easily identify patients with diabetes that are at particular risk of events (information that is crucial to investigators who wish to design clinical trials)? 2. Is fasting glucose concentration independently and convincingly associated with increased risk of cardiovascular events in those without diabetes? 3. Do statins, the most commonly prescribed medications worldwide, have any influence on the risk of developing diabetes? 4. If statins do indeed affect new-onset diabetes, is there any evidence of a dose-dependent effect? 5. How effectively can clinicians predict the development of diabetes in chronic heart failure using commonly recorded clinical information? 6. Does intensive glucose-lowering therapy reduce the risk of cardiovascular events in patients with diabetes? Methods: To address these questions three approaches were used, namely (i) systematic review of previously published data from large cardiovascular endpoint trials conducted in patients with diabetes; (ii) analyses of existing datasets from two large clinical trials; (iii) meta-analyses of published and unpublished data from large clinical trials. Results and interpretation: 1. In a systematic review of 29 trials with 116,790 patients with diabetes, it was apparent that the majority of large cardiovascular endpoint trials conducted in patients with diabetes vastly overestimated the likely cardiovascular event rates in initial power calculations. Introduction of (i) previous history of cardiovascular disease and/or (ii) presence of proteinuria, as binary trial inclusion criteria, provides a simple and effective way to identify patients at high risk, something that is sought after for appropriate clinical trial power calculations. 2. In a population of 6,447 men without diabetes at baseline, impaired fasting glycaemia was not associated with increased risk of cardiovascular events over 15 years. Similarly, when baseline fasting glucose values <7.0mmol/L were split into quintiles, patients in the highest quintile were at similar risk of all vascular endpoints to those in the lowest. By contrast, impaired fasting glycaemia was a powerful risk factor for developing diabetes. 3. A meta-analysis of published and unpublished data from most large placebo- and standard care-controlled statin trials, which included data for 91,140 trial participants without diabetes at baseline, revealed that statin therapy is associated with a 9% higher risk for developing diabetes. 4. A subsequent meta-analysis of unpublished data from five large trials comparing intensive statin therapy with moderate dose therapy found that intensive statin therapy increases the risk of developing diabetes by 12% compared to moderate dosing, in keeping with a dose-dependent effect. While statin therapy remains effective at reducing cardiovascular risk it appears that patients on statin therapy, especially those on intensive regimens, should be considered for diabetes screening. 5. In an analysis of data for 1,620 patients with chronic heart failure and no diabetes at baseline studied for 2.8 years, the strongest predictors of new-onset diabetes were similar to those in the general population. In particular, the combination of HbA1c and body mass index provided a c-statistic of 0.79. 6. In a meta-analysis of published data for 33,040 patients with diabetes who participated in clinical trials comparing intensive glucose-lowering therapy with standard therapy, non-fatal myocardial infarctions were reduced by 17% on intensive therapy but no other cardiovascular endpoints were reduced. Death rates were similar in both groups. Conclusion: While diabetes is associated with excess cardiovascular risk, risk varies considerably depending on other risk factors. Glucose is, at best, a weak risk factor in those without diabetes, and glucose-lowering in patients with diabetes has only yielded a modest reduction in non-fatal myocardial infarctions but not other events; by contrast, measures of glycaemia are powerful predictors of new-onset diabetes in patients with and without chronic heart failure. Finally, the relationship between glucose and vascular disease is further complicated by the fact that numerous medications designed to reduce cardiovascular risk appear to have surprising effects on the risk of developing diabetes.

Recent evidence indicates that immune cells within adipose tissues can drive the formation of ectopic lymphoid structures, known as Fat Associated Lymphoid Clusters (FALC). FALC support B-cell antibody production in response to infection and inflammation. This investigation explores the immune cell composition and role of different adipose tissues both in steady state and during immune responses in mice. Firstly, a detailed analysis of the immune cell composition of peritoneal adipose tissues was performed. To investigate the function and migratory properties of these tissue-resident cells, cytokine and chemokine receptor expression was then assessed. How immune cells in adipose tissues responded to infection was examined using an intestinal helminth infection with the parasite Heligmosomoides polygyrus. Adipose tissues predominantly contained regulatory T cells, invariant natural killer T cells and group 2 Innate Lymphoid Cells (ILC2s). Significant differences were observed in composition, cell surface markers and cytokine production of ILC2s between adipose depots and secondary lymphoid tissues, indicating that tissue specific signals can direct ILC2 responses. Finally, increases were observed in ILC2 and FALC numbers in the mesenteries of WT mice following parasite infection. These data indicate that immune cells within adipose tissues respond to infection and may contribute to immune responses.

The aim of this project was the study of neutrophil recruitment and reverse transmigration using murine and human in vitro models of inflammation. Murine in vitro models of inflammation were developed using an immortalised microvascular cell line (MCEC-1) and primary murine vascular endothelial cells (mEC) isolated from heart and lung. We found that MCEC-1 could recruit murine neutrophils without the requirement of cytokine stimulation, although efficient transmigration did require such a stimulus. Primary cells required cytokine stimulation to recruit mEC. Interestingly, and in contrast to human EC, mEC were relatively insensitive to TNF-α stimulation, although IL-1β was a good stimulus for adhesion and migration. Using the IL-1β driven system we generated reverse migrated murine neutrophils and their phenotype and prolonged survival were assessed. The effect of shear stress and nitric oxide on the regulation of the process of reverse migration was examined. Using adoptive transfer strategies we investigated the fate of mRPMNs in vivo. A significant part of this work involved the study of human reverse migrated neutrophils at a proteomic level using two-Dimensional Fluorescence Gel Electrophoresis methodology to identify changes in neutrophils associated with reverse migration process. We found that murine reverse migrated neutrophils had a very similar surface phenotype to human reverse migrated cells. They also showed prolonged survival. However, our preliminary data on trafficking in vivo did not give a clear indication about their fate upon adoptive transfer into recipient mice. In vitro studies showed that flow generated shear stress and nitric oxide delayed, but did not inhibit, the process of reverse migration. Finally, the proteomics study revealed a number of metabolic, cytoskeletal and regulatory proteins that were differentially expressed in human reverse migrated neutrophils although the functional significance of these changes is yet to be explored.

Patients with rheumatoid arthritis (RA) frequently present with pain under their feet. Forefoot bursae can give rise to such symptoms, but are rarely investigated. The aim of this thesis was to use musculoskeletal ultrasound (MSUS) performed by a podiatrist to evaluate the prevalence and natural history of bursae in the forefoot in RA patients. Once reliability of technique was established, a longitudinal study design was used in which a sample of RA patients (N=149) and a comparator group (N=50) of healthy individuals were assessed at baseline. A Diasus MSUS system was used to image the forefeet of all participants to determine prevalence of bursae. 120 patients (98 female, 22 male) with RA (24 seronegative, 93 seropositive, 3 unknown) completed the study at twelve months: mean age 60.7 (SD 12.1) years and disease duration 12.99 (10.4) years. Results confirmed a high prevalence of forefoot bursae (92.6% of patients; mean per individual =3.54, range 0-9) and that these were often missed by clinical examination. Findings that there could be an association between patient reported foot impact scales of impairment/footwear (LFISIF) and activity participation restriction/limitation (LFISAP) and presence of bursae (LFISIF β=0.377, p=0.033; LFISAP β=0.762, p=0.013) independent of disease activity were unique. On examination of prospective data after one year, 25.8% of participants had increases in bursae and 23.3% decreases. There was a significant correlation between changes in bursae with changes in LFISIF (PCC=0.216, p=0.018) and LFISAP (PCC=0.193, p=0.036) and a significant negative correlation with changes in duration of RA (PCC=-0.269, p=0.003). The findings imply that MSUS detectable bursae in the forefeet are highly prevalent, clinically under-reported and change over time. The findings suggest that bursae within the foot in RA deserve increased clinical attention and that further work is required to confirm associations with patient reported foot impact outcome measures.

No description available.

The potential for nitric oxide (NO) metabolites (e.g. inorganic nitrite) to act as stable stores of “Transported Nitric Oxide” has excited huge interest due to the substantial potential therapeutic avenues. The prospect developing of a “silver bullet” that could target areas most in need of vasodilatation, by releasing NO in areas of hypoxia and ischaemia, could prove a massive advance in the treatment of vascular disease. In this thesis I examine the effects of nitrite infusion in both hypoxia and normoxia. I examine the effects both in health and heart failure, and investigate the potential roles of Nitric Oxide Synthase (NOS) and Xanthine Oxidase (XO) in mediating the reduction of nitrite. We found, and were the first to report in man, that intra-arterial infusions of nitrite had little effect upon the vasculature in high oxygen tension environments but led to significant vasodilatation during hypoxaemia. We found that patients suffering with Chronic Heart Failure responded differently to nitrite infusion to healthy controls, possibly as a result of differences in redox-stress. In healthy volunteers, at rest, neither NOS nor XO appeared to play a significant role in nitrite induced vasodilatation in normoxia and mild hypoxia. We found that vascular myoglobin contributes to the reduction of nitrite to nitric oxide and may play a role in prolonging the vasodilatation induced by nitrite infusion.