Platelets play an important role in the pathogenesis of atherothrombosis and, as such, measuring platelet dysfunction in patients with peripheral arterial disease (PAD) may be a useful prognostic marker for future cardiovascular events. Detecting increased platelet activity but also reduced platelet inhibition is important in the management of these patients. Prediction of cardiovascular risk could allow tailored anti-platelet regimens in high risk groups. Dissection of platelet function in patients with PAD could allow cardiovascular risk stratification and the aim of this thesis was to develop methods to allow 'platelet profiling' in patients with PAD to determine an individual's platelet reactivity. Platelet fibrinogen binding and platelet-leukocyte interactions were determined using flow cytometry. The mechanism of platelet inhibition by nitric oxide (NO) was elucidated further using various inhibitors of the nitric oxide signalling pathway, measurement of cGMP levels and Western blotting for vasodilator stimulated phosphoprotein. In addition, the cytokine profile of these patients was determined, with particular reference to those chemokines important in platelet leukocyte interactions. , Patients with peripheral arterial disease exhibited largely unchanged basal platelet activity (as measured by platelet-leukocyte aggregates and platelet fibrinogen binding), but significantly increased response to the agonist, adenosine diphosphate (ADP). Platelet inhibition in response to NO was reduced in patients with critical limb ischaemia (CLI). In patients with CLI, further work using modulators of the NO pathway demonstrated that the reduced platelet inhibition by NO may be attributable to abnormal protein kinase G (PKG) function. In addition, a global reduction in plasma chemokine levels was seen with increasing disease severity in PAD, including RANTES, MCP-1α and IL-10. This suggests a potential humoral or platelet factor present in patients with PAD causing this change and questions the traditional Th/Th-type cytokine dichotomy in favour of a more integrated cytokine network system. If these observations correlate with clinical outcomes this profiling tool will have important clinical value. This could have therapeutic implications e.g. dual anti-platelet therapy for high risk patients with PAD. Furthermore it would be interesting to study the effect of c1opidogrel and cilostazol or dipyridamole in combined approach to reducing platelet activation (P2Y12 antagonism) and increasing platelet inhibition (phosphodiesterase, PDE inhibition). New therapeutic ideas could be tested, directed by a patients 'profile' in order to combat this platelet hyperactivity in an attempt to reduce the significant cardiovascular morbidity and mortality associated with PAD.

The intermediate-conductance calcium-activated potassium channel, KCa3.1 , allows the efflux of intracellular potassium and is activated by increased intracellular calcium. This plays an important role in vascular physiology and pathophysiology. Expression of KCNN4, the gene encoding this channel, is upregulated in proliferative vascular smooth muscle cells (VSMC) and enhances cell proliferation through increased extracellular calcium influx. Due to its clinical importance, we investigated the regulation of KCNN4 in VSMC. Bioinformatic analysis of the KCNN4 gene was used to determine potential regulatory regions and transcription factor binding sites. Analysis of these sites involved a series of reporter constructs driven by the KCNN4 promoter sequence in promoter deletion assays within both cultured and primary VSMC. This detected potential repressor regions containing putative NFAT, CREB and KLF sites. As both NFAT activity and KCa3.1 expression are perturbed in diabetes mellitus, which is frequently associated with cardiovascular disease, we focused on KCNN4 regulation in response to high extracellular glucose. Whilst KCNN4 mRNA levels were decreased in response to 7.5mM and 11mM glucose, this was not due to NFAT activity at the KCNN4 promoter. In silico identification of CREB and KLF sites within a novel repressor reg ion of the KCNN4 promoter was complemented with electrophoretic mobility shift analysis demonstrating protein-DNA interactions. Mutation of either putative CREB or KLF sites and over-expression of constitutively active or dominant negative CREB proteins had no effect on KCNN4 mRNA levels or promoter activity. However, over-expression of KLF15 reduced both KCNN4 mRNA levels and promoter activity. In addition , stimulation of cAMP production in VSMC caused an 80% reduction in KCNN4 mRNA levels and repressed promoter activity. These data provide insights into the molecular mechanisms controlling KCNN4 expression in VSMC through mechanisms involving KLF15, glucose and cAMP-induced regulatory factors that potentially present novel targets for the therapeutic intervention of vascular 'disease.

Anti-neutrophil cytoplasm antibody (ANCA) associated vasculitis (AAV) is a multisystem disease with substantial morbidity and mortality despite modem treatment. Longer-term patient outcomes are inadequately defined and new safe therapies arc needed. The aims o[this thesis were to investigate long-term outcomes of patients with AAV, compare two different damage assessment scores and the study of a novel immune-suppressant, deoxyspcrgualin (DSG), in the treatment of relapsing and refractory disease. Survival was studied in 535 newly diagnosed patients recruited to four randomised controlled trials. After a median of 5.2 years 25% deaths occurred with a mortality rate ratio of 2.6 (95% Confidence Interval (Cl) 2.2-3.1 ) compared to an age and sex matched general population. Multivariable analysis showed severely impaired kidney function, advancing age, higher disease activity and white cell count and lower haemoglobin were significant negative prognostic factors. In the same cohort, 38% experienced a relapse. Higher risk for relapse was independently associated with anti-proteinase 3 (anti-PR3) antibodies and cardiovascular involvement whereas impaired kidney function conferred a lower risk. A comparison between the Vasculitis Damage Index (VDI) and the Combined Damage Assessment Index (CDA) showed good correlation between both indices. The CDA was more complex but captured more detail. An international expert panel developed recommendations for the conduct of clinical studies and therapeutic trials in AA V. The following areas were covered: disease definitions, disease activity states, outcome measures, eligibi lity criteria, trial design including relevant end-points and biomarkers. Areas for further research were identified. A prospective open label study in 44 patients with refractory or relapsing granulomatosis with polyangiitis (GPA) treated for six months with DSG showed that 95% achieved either partial Of complete remission. Adverse events were common but rarely led to treatment discontinuation. Prolonged administration of DSG in eleven patients was effective III the majority without the occurrence of unexpected toxicity.

Background - Elevation of plasma HDL concentration reduces cardiovascular mortality and morbidity. HDLs have been shown to possess acute anti -inflammatory, anti -oxidant and anti -thrombotic properties. We hypothesise that HDL therapy can acutely alter local and systemic manifestations of plaque instability, Methods and Results - Forty patients with early symptomatic carotid disease were randomised to either receive reconstituted HDL (rI-IDL) - 40mg/kg (11=20), or placebo (11=20). Carotid endarterectomies (CEAs) were performed 24 hours later. Plaques were obtained intra-operatively and used for measurement of thrombomodulatory genes expression. Plasma samples were collected prior to the infusion, 24 and 48 hours later to measure changes in systemic markers of plaque instability. No significant differences were noted in thrombomodulatory genes expression between the two groups. Systemic levels of TF, MMP-9 and MCP-l were significantly reduced in the rHDL group. However, the effects on MMP-9 and MCP-I were abolished in the immediate post-operative period. Although rHDL did not affect plasma IL-6levcls 24 hours following the infusion, it prevented the significant postoperative elevation seen in the placebo group. A similar, but non-significant trend was demonstrated with CRP levels. Conclusion - A single infusion of rHDL can acutely alter plasma biomarkers associated with plaque instability and cardiovascular morbidity.

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Objective: To document the patient characteristics, primary care and hospital management and QoL in patients with intermittent claudication in the United Kingdom. Also to determine the appropriate use of prophylactic therapy, to determine markers of high and low risk and to suggest approaches to optimise the management of peripheral arterial disease. Methods: 474 patients were recruited from 23 centres across the United Kingdom. Data was collected at baseline and at six months and risk factors profile was analysed. Results: Symptomatic disease is more prevalent in men and those above 60. A high proportion of patients were hypertensive and control of blood pressure was not optimal. Use of antiplatelet agents and lipid lowering therapy was less than satisfactory though it improved following hospital referral. Life style modification advice was patchy and not uniform and intensive support for such programmes was lacking. Majority of patients improved not only on clinical parameters but also their QoL over a six month period. 35% underwent peripheral imaging and 7.8% had an interventional procedure. 14% had a vascular event over six months. Low ABPI, high systolic pressure and prior CHD were significantly associated with development of all vascular events. Conclusions: Use of appropriate therapy to reduce the risk factor profile is less than optimal. There is a need for uniform national guidelines for appropriate management of peripheral arterial disease patients in the United Kingdom.

Raised plasma homocysteine (Hcy) levels have been cited as a major risk factor for several vascular disorders. Yet hyperhomocysteinaemia is easily treated through dietary intervention and vitamin supplementation. Commercial assays have facilitated routine plasma Hcy analysis. However, the problem faced by clinicians is stabilisation of Hcy in whole blood samples prior to delivery to the laboratory. Following blood collection, erythrocytes continue to produce and excrete Hcy increasing plasma concentrations by up to 10% per hour. This thesis describes the investigation of stabilising plasma Hcy in whole blood, allowing wide scale screening for hyperhomocysteinaemia. The most effective method appears to be inhibition of the enzyme responsible for Hcy production, Sadenosylhomocysteine hydrolase (SAHH), using a competitive inhibitor 3- deazaadenosine (3DA). Clinical trials were conducted on a pilot batch of evacuated blood tubes. Samples were stored in EDTA whole blood in the presence and absence of 3DA, at ambient temperatures (20 to 25ºC), and under refrigerated conditions (2 to 8ºC). Only samples that were collected into EDTA plus 3DA tubes and stored refrigerated showed stability over 72 hours (p = 0.2761). For wide scale screening, samples must be stable under ambient conditions. As the structure of SAHH is known a molecular modelling approach was adopted in an attempt to identify other potential inhibitors from screened databases. Interference of SAHH, in an immunochemical method for Hcy, was to be utilised for in vitro screening before any further clinical trials were conducted. The thesis also focuses on Hcy as a marker of vitamin deficiency and explores links between thiol metabolism and the development of cognitive decline eventually leading to dementia. Disruption of single carbon metabolism can lead to an increase in Hcy and a decrease in available methyl groups important in regulation of several metabolic pathways. Increased oxidative stress may also be a causative factor.

Previous work has established that Eph family receptor tyrosine kinases and ephrin ligands control a wide range of morphogenetic processes in vertebrate embryos through cell-contact dependent signalling interactions. In the developing cardiovascular system, ephrin-B2, a transmembrane protein is expressed by arterial endothelial cells (ECs) whereas the cognate receptor EphB4 is predominantly found on the venous endothelium. Gene targeting studies in mice have demonstrated that both molecules are critically required for angiogenic remodelling of embryonic blood vessels and survival beyond midgestation. To gain more insight into the role of ephrin-B2 in vascular development and its arterial expression, I have used the tetracycline-controlled expression systems to overexpress the ligand in the endothelium of all vascular beds (i.e. in arteries, veins and microvessels) of transgenic mice. In the course of this study, I have employed several different transgenic EC-specific driver lines in combination with tetracycline-controlled (tTA, Tet-OFF) and reverse tetracycline-controlled (rtTA, Tet-ON) transactivators. Ephrin-B2 overexpression triggers enhanced activation of EphB receptors particularly in the venous endothelium. This leads to severe vascular malformations such as oedema and haemorrhaging. Induction of ephrin-B2 expression at different stages of embryonic development controls not only vascular patterning and the recruitment of supporting pericytes and vascular smooth muscle cells but it can also trigger tissue-specific responses. In summary, my work has established that ephrin-B2 is an important regulator of blood vessel morphogenesis throughout embryonic development. Some results suggest that the ligand may also be involved in pathological conditions such as fibrosis as ectopic expression of ephrin-B2 in the embryonic liver triggers the activation of hepatic stellate cells. The resulting increase in matrix deposition around hepatic blood vessels could represent early signs of a fibrotic phenotype.

Analysis of blood pressure and flow waveforms may lead to improved diagnosis of arterial dysfunction and disease. This thesis describes experiments to investigate the characteristic alteration of peripheral waveforms produced by stimulated release of vascular nitric oxide (an effect that is attenuated by endothelial dysfunction) and the changes that occur with age. In vivo experiments were conducted in anaesthetised rabbits and in vitro experiments employed a polyurethane model of the human aorta and its principal branches. Blood pressure, blood flow, pulse wave velocities and vessel diameter were recorded in the rabbit abdominal aorta. Equivalent recordings were obtained from the model aorta. Data were analysed in the time domain using wave intensity analysis after separation of reservoir (Windkessel) pressure and wave pressure. Effects of acetylcholine (Ach) and NG-nitro-L-arginine methyl ester (L-NAME) were investigated in vivo. Ach (which promotes endothelial production of nitric oxide) increased wave reflection, whilst L-NAME (which inhibits nitric oxide production) decreased it. These trends were opposite to the expected ones, and do not account for the established effects of nitric oxide on peripheral arterial waveforms. Further work is required to investigate these contradictions. Arterial stiffening in immature and mature rabbits was attempted by supplementing their diet with fructose. Fructose is known to form advanced glycation end-products in arterial walls and hence to stiffen arteries. Unexpectedly, fructose did not affect haemodynamic function. However, immature control rabbits had markedly reduced aortic wave reflection compared to mature control rabbits, indicating that the former have arterial impedances that are better matched for incident wave propagation. Wrapping the model aorta in Clingfilm was used to simulate age- or drug-induced stiffening of the aorta in vivo. Increased pulse pressure was observed, resembling the isolated systolic hypertension prevalent in aged populations. The model has potential for modelling haemodynamic function in health and disease.