The work reported in this thesis is mainly biochemical research on the properties of the human immune system plasma protein, complement factor H (FH). A major function of FH is to bind to “foreign” or “altered-host” surfaces, mainly by recognising charge cluster motifs. When bound to a surface, it down regulates activation of the complement system on that surface. Its binding properties towards a range of other proteins and macromolecules, have been examined, mainly by ELISA-style assays. Research then focused on a smaller number of these FH ligands, which appear to bind FH very strongly, and not, as is the usual situation, by charge interactions. These ligands are Adrenomedullin, Trinitrophenyl-derivatised ligands, and dinitrophenol-derivatised ligands (TNP and DNP). The binding and dissociation characteristics of these ligands have been examined, the binding optimised, and it has been shown that TNP and DNP derivatised ligands can be used for affinity purification of FH from human plasma. A factor H homologue in plasma, C4bp, also binds these ligands, but a number of other FH homologues in plasma do not (eg beta2 glycoprotein1). Expression systems have been obtained from other labs to make recombinant segments of FH and recombinant protein expressed in order to narrow down the binding sites on FH for these ligands. Binding sites in 3 regions of FH have been located, and the effects of ligand binding on the complement-regulatory functions of FH have been assessed.

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This thesis is concerned with the renin-angiotensin system in essential hypertension. Humoral agents have been sought in the causation of high blood pressure for many years. The role of the renin- angiotensin system in hypertension remained controversial, but, gradually, the components and functions of this complex hormonal system were elucidated. Derangements of the system were confirmed in various examples of secondary hypertension, but the role of the renin-angiotensin system in essential hypertension remains unclear. The belief that essential hypertension represents an homogeneous group of patients has been questioned in recent years, and this has led to the attempt to separate essential hypertensive patients into renin subgroups. One of the most intensively investigated has been that with low-renin, but whether or not this group represents a distinct entity is uncertain. An important hypothesis attributes this 'syndrome 1 to an excess of an unknown mineralocorticoid. This work was undertaken to reappraise the role of renin in essential hypertension. Two major questions were considered: Is the activity of the renin-angiotensin system similar in essential hypertension and in the general population? Does low-renin hypertension exist as a separate entity attributable to mineralocorticoid excess? In answering these questions many variables other than renin, including age, sex, blood pressure, sodium, potassium, plasma volume and aldosterone, and their interrelationships were considered. Plasma renin activity is an index of the activity of the renin-angiotensin system, and the method used in its measurement has been described in considerable detail. Volunteers to have their arterial pressure measured were sought amongst the employees of A.E.R.E. Harwell and two general practices in Wantage and Didcot. From them two samples of people willing to co-operate were chosen: 89 with arterial pressures above 100 mm Hg and 89 of similar age and sex with diastolics below 90 mm Hg. All subjects with high blood pressure who had received treatment or who had symptoms from it were excluded. Investigations showed that subjects with high pressures conformed to the diagnosis of essential hypertension. RENIN IN ESSENTIAL HYPERTENSION Blood was withdrawn for the measurement of plasma renin activity after the subject had been lying down for 2 hours (supine) r after being up and around for 2 hours (erect), and 1 hour after an intravenous injection of frusemide (1 mg/kg). The renin responsiveness or change in plasma renin activity after stimulation was expressed in absolute and in percentage terms. In essential hypertension plasma renin and its responsiveness were found to be suppressed compared to control subjects. Reasons for the reduction in plasma renin and in renin responsiveness in essential hypertension are uncertain, but various factors are known to affect renin and were considered Plasma, urinary and total body potassium, plasma, urinary and total exchangeable sodium, and plasma volume were studied but did not account for the difference. Age was shown to correlate inversely with renin and its responsiveness in patients with hypertension and in controls, but the groups were age matched. The effects of previous antihypertensive drugs on renin levels were excluded by rejecting previously treated patients. Females tended to have lower plasma renin activity and responsiveness than males, but those with hypertension were sex matched with the controls. However, blood pressure was shown to be inversely related to plasma renin and its responsiveness in both groups. Thus, reduced plasma renin activity and responsiveness appears to be a general feature of essential hypertension. The suppression of renin is probably a result of the combined effect of many factors on renin release, but blood pressure itself has been shown to be an important contributor. LOW-RENIN HYPERTENSION An analysis of the distributions of plasma renin and renin responsiveness showed the logarithms of the renin variables to be normally distributed with no evidence of bimodality. This suggests that the low-renin state forms part of a continuum in hypertension rather than a distinct diagnostic entity. An attempt was made to separate the subjects with high arterial pressure into renin subgroups in a statistically acceptable way, while utilizing basal, stimulated and renin responsiveness data. The distributions of plasma renin and renin responsiveness were divided into three equal subgroups (lower, mid and upper) on the understanding that if a low- renin subgroup existed it would be contained in the lower third of the distributions. Plasma renin correlated significantly with responsiveness; in view of our method of defining low-renin hypertension, the decreased responsiveness in the low-renin subgroup could not be used to support its existence as a separate entity. This strengthens the argument against the use of stimuli to define a low-renin subgroup in essential hypertension. The hypothesis that an unknown mineralocorticoid is involved in the aetiology of low-renin hypertension did not withstand critical examination. Total exchangeable sodium, plasma volume, total body potassium and 24 hour urinary potassium excretion were measured in the three renin sub- groups of essential hypertension; the characteristic findings of an excess of an aldosterone-like mineralocorticoid were absent. It is proposed that low-renin hypertension represents no more than the lower end of the normal distribution of renin in essential hypertension. However, many factors including age and blood pressure may modify the renin status and need to be taken into account in the interpretation of individual values.

Endothelial microparticles (EMPs) are complex structures with pleiotropic properties and are emerging as an index of endothelial damage. Increased circulating levels of EMPs have been identified in several inflammatory disorders and are reduced following anti-inflammatory treatment. Since they are cell-to-cell communicators, this study aimed to identify specific effects on both endothelial and vascular smooth muscle cell (VSMC) function. We hypothesise that EMPs have a dual role, depending on the stimuli involved in their release, potentially playing a role in vascular homeostasis, but also in exacerbating vascular damage under disease conditions and this can be executed via activating the endothelium, and also in the cross-talk to the smooth muscle layer, in terms of depositing a calcified matrix. The study is presented in two sections: first, to investigate the effect of EMPs on endothelial cells, by studying EMP release, endothelial cell activation and migration, and second, to investigate whether they modulate osteogenic differentiation of VSMCs in vitro, focusing on the mechanistic pathways involved using a microRNA and proteomic screening. To achieve these aims, three different sets of extracellular vesicles were generated (probably containing EMPs and exosomes): i) uEMPs, which were generated from healthy growing untreated human umbilical vein endothelial cells (HUVECs), ii) sEMPs, from Tumor Necrosis Factor alpha (TNFα)- stimulated HUVECs and iii) AoEMPs, from TNFα-stimulated human aortic endothelial cells. In the first study, HUVECs were treated with either uEMPs or sEMPs for 24 hours to investigate their effects on endothelial cell function, while in the second study, VSMCs were treated with AoEMPs for 3 weeks in osteogenic media to assess their effects on vascular calcification. The study confirmed that EMP content, which depends on their cellular origin and the stimuli involved in their release, defines their properties. Both uEMPs and sEMPs increase vascular cell adhesion protein (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) and cell migration. However, sEMPs increase EMP release and carry elevated Chemokine (C-C motif) Ligand 20 (CCL20), identified in the proteomic screening and validated by flow cytometry, in comparison to uEMPs, thus contributing to the elevated EMP levels and disease pathogenesis via CCL20 and dysregulated inflammatory pathways. In addition, Alizarin Red S and calcium deposition assays demonstrated that VSMCs treated with AoEMPs in osteogenic media for 3 weeks show enhanced calcification in vitro, using Alizarin Red staining and calcium deposition assays. These findings may be in part, linked with miRNA- 3148/osteoprotegerin signalling pathway, as miRNA-3148 was identified in the microRNA screening and using transfection studies, we identified its relationship with its target osteoprotegerin RNA. This study provides improved understanding of the mechanisms in which EMPs affect endothelial function and VSMC calcification in vitro. Further research will help understanding the ultimate role of EMPs on the vessel wall.

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Detailed knowledge about the mechanisms involved in vascular establishment, differentiation and growth is essential to develop new therapies to treat blood vessel formation related conditions like cancer and blinding eye diseases. Although research has investigated the cellular mechanisms of vessel growth in great detail, a clear understanding of the integration of extracellular signals into the transcriptional programme regulating blood vessel formation still remains elusive. In this thesis the role of the transcription factor Tal1 as a regulator of blood vessel formation and differentiation is investigated. Tal1 is a master regulator of embryonic haematopoiesis, adult megakaryopoiesis, and erythrocyte maturation. Tal1 depleted mice die at gestational day E9.5 due to a complete lack of blood. However, several lines of evidence also suggest an important function of Tal1 in blood vessel development. I chose an inducible, endothelial-specific knock-out strategy to investigate the effects of Tal1 depletion in mice with a focus on vascular development. A distinct time window during embryogenesis, in which Tal1 is required for the proper formation of the primitive intra-embryonic vascular network and the dorsal aorta, was identified. In contrast, I demonstrate that there is no requirement of Tal1 for postnatal sprouting angiogenesis or blood vessel homeostasis. This suggests an important role in vasculogenesis rather than angiogenesis and is in line with recent findings which attribute the proper differentiation of endothelial cells to Tal1. In addition, I have generated and characterised transgenic mouse lines in which Cre recombinase activity is controlled by a Dll4 promoter/enhancer construct. Thereby, Cre expression is specifically directed to the arterial endothelium. I believe that this model can be of great interest for angiogenesis research in general and especially for the understanding of induction and maintenance of arterial-venous identity.