Structural information for the zebrafish P2X4 receptor in both an agonist bound and unbound resting state provided a major advance in understanding agonist action and has given insight into movement that occurs in the receptor upon ATP binding. Despite agonist action now being well characterised, the molecular basis of antagonism is poorly understood. In this thesis the mechanism of antagonist action at the hP2X1 receptor has been investigated through determining properties of chimeras and mutant receptors based on differences between antagonist sensitive and insensitive P2X receptors. The antagonists suramin, NF449 and PPADS potently inhibit the human P2X1 receptor but have little or no action at the rat P2X4 receptor. The extracellular loop of the hP2X1 receptor was shown to determine antagonist sensitivity and was therefore split into four sections, residues of which were swapped with corresponding residues of the antagonist insensitive rP2X4 receptor and vice versa. Sub-chimeras and point mutations were then made to identify particular residues and regions which contribute to antagonist action. These experiments identified two regions important for NF449 binding at the receptor. These are a cluster of four positively charged residues at the base of the cysteine rich head region (136-140) and three residues located just below them (T216, H224 and Q231). An NF449 bound model of the hP2X1 receptor has been generated. The introduction of the four positively charged residues at the base of the cysteine rich head region to the rP2X4 receptor introduced suramin and PPADS sensitivity to this previously insensitive receptor. This mutation is thought to cause a conformational change which allows the antagonist to bind at residues which are already present in the wildtype receptor. In summary this thesis has advanced the understanding of antagonist action at the hP2X1 receptor and the antagonist insensitivity of the rP2X4 receptor.

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Background: Currently available prosthetic small calibre vascular grafts have poor medium and long term outcomes due to the development of neo-intimal hyperplasia caused by their non-compliant properties and lack of an endothelial cell lining. Here a nanocomposite based on polyhedral oligomeric silsesquioxane (POSS) attached by direct reaction onto a urethane segment was employed as a potential vascular graft material. It has been demonstrated to have similar viscoelastic properties to a native artery and be resistant to degradation. Initially Human Umbilical Vein Endothelial Cells (HUVEC) were employed as the cell source of choice after which investigations were carried out into the suitability of human peripheral blood derived circulating endothelial cells (CEC) and endothelial progenitor cells (EPC) for graft seeding. Aim: The aim of this study was to develop a system with the potential to deliver an EPC/CEC-seeded bypass graft in a realistic time-frame. Methods: The cytocompatibility of the nanocomposite was initially investigated using HUVEC as a cell source. Surface modification of the nanocomposite to improve cell adhesion and proliferation was then attempted using UV exposure. Seeded nanocomposite grafts were then exposed to flow and the effect of preconditioning investigated. Following this the use of human peripheral blood derived EPC and CEC as a potential cell seeding source was investigated. Studies were also carried out into the sterilization of the nanocomposite. Results: The nanocomposite was able to support the attachment and growth of both HUVEC and EPC/CEC. Nanocomposite grafts were successfully seeded and exposed to flow with cells being retained on the graft surface following exposure to flow. Conclusions: The results obtained suggest that the nanocomposite graft and the use of EPC/CEC derived from human peripheral blood process has potential both for a realistic and achievable two-stage seeding process for vascular bypass grafts.

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No description available.

Oligosaccharides of hyaluronan (o-HA; 3-10 disaccharides) are pro-angiogenic and are believed to act through two receptors, C044 and RHAMM (receptor for hyluronanmediated motility). Utilizing human liver micro-vessel-derived endothelial cells (HLMVEC)and bovine aortic endothelial cells (BAEC), the roles of C044 and RHAMM in o-HA induced signalling were examined. In vitro assays were employed for proliferation, migration and tube formati~m by endothelial cells, prior to and following C044 and RHAMM knock-down, using small interference RNA (siRNA) technology. Epidermal growth factor (EGF), which has stimulatory effect on endothelial cells, but does not act through C044 or RHAMM, was used to assess specificity of the results. All data were analysed using SSP statistical package and p values S 0.05 were considered significant. The results are summarised as follows: • Following knock-down of CD44 and RIIAMM, using siRNA technology, in HLMVEC and BAEC, the result of RT-PCR and Western blotting revealed a highly significant decrease in their gene and protein expression in both cell types. • A significant increase in cell proliferation, as quantified by cell number, was obtained in the presence of o-HA and EGF, compared with untreated controls. • Knock-down of RIIAMM, significantly inhibited BAEC (-30%) and Hr_.MVEC (-25%), whereas in neither cell type knock-down of C044 had no effect on cell proliferation in either cell type. RHAMM and CD44 knock-down almost completely inhibited o-HA-induced cell proliferation, compared with controls and negative control (NC) siRNA-treated cells. In contrast, an angiogenic growth factor, epidermal growth factor (EGF) which operates independently of CD44 or RHAMM was used as a control. After knocking-down C044 and RHAMM in BAEC or HLMVEC, EGF induced proliferation remained unaffected compared with, controls • In wound healing migration assay, treatment with o-HA significantly stimulated migration of BAEC compared with controls. Following knock-down of CD44 and RHAMM, o-HA-induced wound healing was markedly decreased compared with controls. • In another assay for migration that utilizes Boyden chamber, again o-HA and EGF significantly promoted migration of HLMVEC. Following knock-down of RHAMM and CD44, o-HA, unlike EGF, induced significantly lower (-5-fold) migration compared with controls. • In Matrigel assay for tube (capillary net-work) formation, o-HA and EGF significantly enhanced tube formation by BAEC and HLMVEC. Knock-down of CD44 even in the absence of o-HA induced a significant increase in tube formation compared with non-transfected cells. In o-HA stimulated BAEC and HLMVEC tube formation significantly decreased in comparison to controls, but the effect of EGF remained the same in knock-down and controls. • To ascertain possible signal transduction mechanisms, it was shown that CD44 and RHAMM acted through phosphor-ERK expression in o-HA treated cells, compared with control cells. In both BAEC and HLMVEC. a significant increase was observed in phospho-ERK1I2, compared with the controls. After knocking down CD44 or RHAMM there was a significant decrease in phospho-ERK1I2 expression, compared with untreated controls, in both BAEC and HLMVEC. Collectively, these results suggest that o-HA induced signalling pathways are independently mediated by RHAMM and CD44 receptors in micro- and macro-vascular EC, through a mechanism that remains to be fully elucidated

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The development of an artificial bypass graft for the treatment of peripheral vascular disease would overcome the limitations of using autologous veins. An ideal graft would be a tubular structure made from an elastomeric polymer. Layers of autologous cells would then be cultured on the inside of the tube in order to render the graft nonthrombogenic. The aim of this work was to modify the surface of a polyurethane by covalently attaching recombinant extracellular matrix proteins in order to gain a degree of control over the behaviour of smooth muscle cells. A chemical modification protocol was used to attach the proteins to the polyurethane via a layer of dextran. The proteins used were an RGD-containing fragment of fibrillin-l and full length tropoelastin. These are proteins found in the artery wall. Protein imrnobilisation was verified using Enzyme Linked Imrnunosorbent Assays. RGD-dependence of cell attachment was investigated by incubating the cells with a soluble RGD-containing peptide prior to seeding. The contribution of integrins a vP3 and aSPI to the attachment of the cells was determined using blocking antibodies. Cell proliferation and spreading were quantified. Gene expression and protein production were investigated using RT-PCR, imrnunostaining and Western blotting.

No description available.