Previous work, on RyR1/2 modulation, assessing phosphorylation in relation to FKBP12/12.6 stabilisation of channel activity, indicates convergent regulation involving control of domain interaction sites by phosphorylation, FKBP12/12.6 and other modulators. This study used a central domain peptide (DP4WT) and its mutant form (DP4M) to investigate the relationship between FKBP12, phosphorylation, and other modulators to identify this region of convergent regulation. Native RyR1 channels, stripped or endogenous FKBP12 and phosphorylated or dephosphorylated, were shown, by [<super>3</super>H]ryanodine binding, to be fully functional and to be activated by calcium and ATP. FKBP12 and magnesium were each shown to inhibit channel activity, and phosphorylation reversed magnesium inhibition. DP4WT peptide, synthesised from two sources, activated RyR1 2-fold in the presence of &micro;M calcium, less than activity observed with ATP. Magnesium and FKBP12 inhibited RyR1 activation by DP4WT. In contrast to previous studies, DP4M was not an inactive control peptide. To investigate this discrepancy, recombinant GST-tagged (R-)DP4M and (R-)DP4WT were generated and neither showed an activatory effect. However, once cleaved of GST, the R-DP4WT was as potent as synthetic DP4WT at 10 &micro;M. Experiments with RyR2 demonstrated similar results for calcium and ATP activation with inhibition by FKBP12.6. The RyR2 central domain peptide (DPc10) has been cloned and the protein produced.

The adenosine A3 receptor is one of the suspected effectors of intrinsic preconditioning. Its intrinsic ligand, adenosine is produced by ischaemic tissue, where it acts to cause a preconditioning effect, rendering the tissue resistant to damage by any subsequent ischaemia. Adenosine A3 receptor stimulation has previously been shown mediate the recovery of the heart following ischaemia. It is thought to be involved in alleviating temporary stunning and modulating infarction by the mechanisms by which it evokes preconditioning. Using a variety of cardiac ischaemia models from rat tissue, we investigated the effect of a selective A3 receptor agonist on ischaemic cardiac tissues and whether it is possible to evoke a cardioprotective effect by stimulation of adenosine A3 receptor alone. The administration of selective A3 receptor agonist alleviated post-ischaemic contractile dysfunction in the isolated left atria. No effect was found in right ventricle strips, while in the Langendorff heart it was found that there was a pro-infarct effect, with no observed effect of contractility. Attempts to classify the effects as being adenosine A3 receptor -mediated or another adenosine subtype by use of selective antagonists was difficult for reasons discussed. Using immunological methods is was possible to identify a protein specific for the adenosine A3 receptor in membrane fractions obtained from both normoxic and ischaemic rat left atria. This strengthens the claim that the effect reported in this thesis are adenosine A3 receptor mediated. In summary it is recommended that A3 receptor manipulation and its effect on post-ischaemic recovery remains a viable route of further investigation. The work in this field is required to form new strategies for the treatment coronary heart disease and other cardiac conditions.

Whilst evidence from human studies suggests that complement activation is pro-atherogenic, studies using animals models of the disease, including the low density receptor deficient (ldlr<super>-/-</super>) and apolipoprotein E deficient (apoE<super>-/-</super>) mouse models, contradict one another. The hypothesis underpinning this thesis is that the complement system contributes to disease pathology in atherosclerotic plaques of apoE<super>-/-</super> mice. The work focussed on the membrane attack complex (MAC) of the terminal pathway and the central component of the complement system, C3. I have shown that in the absence of the MAC regulator CD59a, apoE<super>-/-</super> mice had accelerated atherosclerosis compared to controls, accompanied by increased MAC activation within the plaques. In accordance, C5 deficiency was protective against atherosclerosis in apoE<super>-/-</super> mice, a result of absence of MAC in these mice. However, MAC inhibition using an anti-C5 antibody in apoE<super>-/-</super> mice did not inhibit progression of atherosclerosis. Surprisingly, in the absence of CD55, apoE<super>-/-</super> mice had smaller atherosclerotic lesions together with an anti-atherogenic lipoprotein profile and increased C3 activation product, C3adesArg, in their plasma. The data reveal a novel role for CD55 during lipid metabolism and, together with published data on the metabolic role of C3adesArg, highlight the need for further investigations into the role of complement during lipid metabolism.

No description available.

Interdomain Interactions within the complex three-dimensional architecture of the cardiac ryanodine receptor (RyR2) are pivotal in channel regulation. Acquired or genetic abnormalities that perturb these stabilising intra-molecular interactions are pathogenic. This laboratory identified the interacting- or l-Domain of human RyR2 that mediated interaction between cytoplasmic and transmembrane (TM) assemblies. To further elucidate the precise roles of functional motifs within the l-Domain, three contiguous fragments spanning RyR2 amino acid residues 3722-4610 were synthesised using a cell-free system. One fragment termed IDB (amino acid residues 4353-4499) profoundly modulated cellular Ca2+ cycling and resulted in the remarkable normalisation of intercellular synchrony following its microinjection into ouabain-treated cardiomyocyte monolayers. These phenomena were linked to IDB- mediated stabilisation of RyR2 and were fully corroborated using IDB purified from a bacterial expression system. Bioinformatic analysis revealed striking structural homology between sub-fragments of the RyR2 l-Domain and l-Domain-like regions of inositol 1,4,5- trisphosphate receptors (IP3R). Recombinant expression of l-domain sub-fragments in RyR- null human embryonic kidney (HEK) cells remodelled carbachol-evoked Ca2+-responses and suppressed homeostatic Ca2+ signalling events indicating that IDB also modulated IP3R signalling mechanisms. In both HL-1 and HEK cells, IDB-dependent Ca2+ modulation extended to surrounding cells that were not microinjected with recombinant protein. This so- called 'bystander effect' was mediated by the transfer of signalling molecules via direct cell- to-cell coupling (gap junctions) and also by the extracellular transmission of diffusible effectors. This thesis supports the concept that RyR2 stabilisation rescues pathogenic Ca2+ dysregulation and suggests that there is substantial merit in developing further epitope-targeting strategies for the therapeutic normalisation of Ca2+ cycling in cardiac disease.

These findings suggest that systemic and vascular wall levels of matrix metalloproteinase-9, related to inflammation at the joint site, may play a prominent role in the development of vascular dysfunction in this experimental model. This thesis goes someway to elucidating the potential mechanisms of vascular dysfunction in rheumatoid arthritis.

Introduction: The purpose of the project is to simulate the effect of hypothetical intervention on risk of vascular disease in the Caerphilly cohort. The cohort comprises a total population sample of 2959 men aged 45--59 years at the recruitment who has been followed up for 20 years. During that time there has been particular emphasis on assessing exposure to vascular risk factors and assessing vascular related outcomes. Aim: The aim of the thesis is to estimate the effects at population level of public health interventions to change the levels of modifiable risk factors for the vascular disease. Methods: Various statistical techniques such as logistic, fractional polynomial and Cox's proportional hazards models along with various parametric models were used to analyse the data. New risk prediction models were estimated and compared with the existing models in the literature. Various standard simulation techniques were used to simulate hypothetical data using Caerphilly data parameters. Hypothetical interventions were carried out on these generated samples to assess the public health impact. Results: Multivariate analysis suggested that the combined effect of psychological variables measured in the study were significantly associated with the increased risk of MI. New risk prediction models constructed using the Caerphilly study data showed that they were significantly different from the standard available models from the literature. Simulation results suggested that there could be a reduction MI events by 25--30% and stroke events by 50--55% using plausible intervention scenarios available from the literature review. Conclusion: A hypothetical intervention to modify psychological factors showed a higher reduction in MI events. Therefore, plausible interventions to modify psychological factors should be commissioned along with the standard biological and behavioural interventions.

No description available.

In cardiovascular disease, the deleterious effects of Angiotensin II (Ang II) on the vasculature are well recognised. This is not necessarily due to elevated levels of circulating Ang II. An alternative hypothesis implicates the role of vascular cell signalling in disease. Therefore this thesis examined the role of the Ang II type I (AT1) receptor in a cell culture model of hypertension: cultured human vascular smooth muscle (VSM) cells were exposed to cyclical mechanical strain regimes (Flexcell®) designed to mimic the forces generated by hypertension in vivo. Early experimentation demonstrated cyclical mechanical strain and Ang II induction of VSM cell gene expression and synthesis of extracellular matrix proteins. Importantly, AT1 receptor antagonism inhibited the strain-induced fibrogenic activity of VSM cells suggesting a synergistic relationship between the renin-angiotensin system and mechanical strain. The response of VSM cell AT1 receptor to mechanical strain was analysed by three-colour flow cytometry. After accounting for non-specific binding, two distinct populations of human VSM cells were identified based on their level of AT1 receptor expression. In the population of VSM cells with a high expression of AT1 receptor, cyclical mechanical strain resulted in an increase in the expression of the AT1 receptors. This thesis has revealed for the first time, conclusive evidence that mechanical strain up-regulates cell-surface expression of AT1 receptors on human VSM cells. This may highlight a mechanism whereby mechanical strain may lead to sensitisation of human VSM cells to Ang II in the early stages of vascular disease.

Background: Epidemiological studies lend some support for a genetic predisposition to human stroke. There is a growing body of evidence to suggest a role for Angiotensin II (ANGII) in vascular disease. The levels of Angiotensin converting enzyme (ACE), which converts ANGI to ANGII, are known to be under a significant degree of genetic control. The D allele of the ACE I/D polymorphism is associated with higher serum ACE levels and this allele has also been associated with ischaemic stroke. Recent identification of numerous ACE single nucleotide polymorphisms has allowed for a more powerful case control haplotype analysis of ACE in ischaemic stroke. Patients and Methods: The validity of the published structure of the common ACE haplotypes was investigated and supported using long range allele specific PCR and DNA sequencing of a random sample of UK caucasian subjects. Using restriction fragment length polymorphism (RFLP) analysis of selected polymorphisms we generated ACE haplotypes for 359 ischaemic stroke patients and 328 unrelated controls. Results: Age, hypertension, smoking, diabetes and hypercholesterolaemia were identified as significant clinical risk factors for ischaemic stroke. D allele frequencies showed no significant differences between cases and controls (0.55 vs 0.53 respectively). However a low frequency D allele haplotype (H9) was found to be an independent risk factor for ischaemic stroke (odds ratio 2.05, p=0.004). Conclusion: This study has provided allele specific data to support the haplotype structure of the common ACE haplotypes in a UK caucasian population. For the first time, in a case control analysis, we have identified a significant and independent genetic association of a low frequency ACE haplotype, H9, with human ischaemic stroke. This result suggests an important role for ACE in ischaemic stroke which will require further study in other populations using a variety of control groups.