The molecular identity of the KATP channel of vascular smooth muscle was investigated using a variety of methods. RT-PCR was used to investigate the expression of KATP subunit mRNA. Kir6.1, 6.2, and SUR 2B transcripts were detected in all vascular smooth muscle preparations, SUR 1 was detected in mesenteric artery. Expression at the protein level was determined, in femoral artery cells, using an immunohistochemical technique, visualizing stained cells with confocal microscopy. Antibodies specific for Kir 6.1, 6.2 and SUR 2B, 2A, subunits were used and all resulted in staining. Using a Xenopus expression system cloned KATP channel currents were characterized. Differences in channel activation, via metabolic poisoning, were found to be dependent on the SUR subunit. The compound PNU-37883A was found to be selective for the Kir subunit, with channels comprising Kir 6.1 being more sensitive to inhibition by this compound than Kir 6.2, Kir 6.x chimeras were constructed to identify a possible site of modulation/binding and a region of the C-terminus was identified which was important in the channels response to the compound. The differences in sensitivity of channels with Kir 6.1 and 6.2 are similar to those reported for vascular smooth muscle and skeletal or cardiac muscle, providing further evidence that the Kir 6.1 subunit is involved in forming the KATP channel of vascular smooth muscle. However, the other evidence provided here doesn't rule out the possibility of heteromultimeric KATP channel formation in vascular smooth muscle.

The regulation of microvascular survival impacts both developmental remodelling of the vasculature, and various pathologies. The aim of this thesis was to examine the role of a recently identified A20 binding inhibitor of NF-kB, a zinc finger protein termed ABIN-2 in endothelial protection. More specifically, looking at its effects on inflammation and apoptosis in endothelia both at the cellular level and in vivo. The involvement of ABIN-2 in the Tie2 receptor pathway was also examined. Tie2 is an endothelial receptor essential for blood vessel formation and promotes endothelial survival. A transfection protocol was established allowing expression ABIN-2 in up to 90% of endothelial cells. ABIN-2 was shown to reduce apoptosis in endothelial cells as well as improve cell survival following growth factor deprivation. This effect was inhibited by Wortmannin and LY294002 which are known inhibitors of phatidylinositol-3 kinase. Expression of the truncated form of ABIN-2 lacking the carboxy terminal of ABIN-2 did not protect cells from apoptosis. In addition, expression of the truncated form prevented cell rescue by angiopoeitin-1 from apoptosis. The chick chorioallantoic membrane was used as an in vivo model for testing the role of ABIN-2 in vessel inflammation. It was possible to use this model to transfect plasmids into live microvessels using electroporation resulting in high yield expression of target protein. This model was adapted to look at microvessel inflammation and apoptosis. Expression of ABIN-2 in microvessels reduced leukocyte rolling following tumour necrosis factor-a and lipopolysaccharide induced inflammation. The truncated form of ABIN-2 lacking the carboxy terminal did not reduce microvessel inflammation. The NF-kB inhibitor PTDC was found to suppress leukocyte rolling. ABIN-2 expression also appeared to give limited protection against apoptosis in vivo.

Advanced Glycation Endproducts (AGEs), resulting from the non-enzymatic reaction of reducing sugars with proteins, accumulate in patients with diabetes mellitus and with advancing age and are implicated in the pathogenesis of vascular disease. Oxidative stress also participates in vascular pathology and has also been reported in the context of diabetes and ageing. This study set out to explore the contribution of AGEs to oxidant stress generation, particularly by examining their effects on the respiratory burst of neutrophils and lymphoblasts. Using chemiluminescence to detect reactive oxygen species (ROS), AGEs did not stimulate the neutrophil respiratory burst directly, but caused a dose-dependent enhancement of the neutrophil respiratory burst in response to a mechanical stimulus (up to 265% +/- 42%, p=0.022) or chemical stimulation with fMLP (formylleucylphenylalanine) 100nM (up to 218% +/- 19%, p<0.001). This phenomenon was immediate and reversible, and depended on the simultaneous presence of AGEs with the additional stimulus; hence AGEs appear to act as neutrophil 'co-agonists'. The in vivo correlates of mechanical and chemical stimulation may be vascular stress and microbial exposure respectively, especially since some acute vascular events have been correlated with infective episodes. The 'co-agonist' effect of AGEs on the neutrophil respiratory burst appears to involve upregulation of the NADPH oxidase enzyme, as evidenced by a DPI-dependent suppression of basal and augmented ROS output. This in turn is dependent upon the generation of arachidonic acid (which may potentiate NADPH oxidase subunit function), via cytosolic phospholipase A2 (cPLA2) activation. The whole process is sensitive to adjustments of the intracellular redox status, implying a role for upstream redox signalling.

No description available.

The blood clot is composed of a mesh of fibrin fibres with cellular elements embedded in this network. The structure of the fibrin clot can determine predisposition to atherothrombotic conditions, as compact clots composed of thin fibres and small pores are associated with premature and more severe cardiovascular disease. Both genetic and environmental effects can cause qualitative and quantitative changes in procoagulant and anti-fibrinolytic proteins, altering fibrin clot formation, structure and lysis. Three hypothesese were tested in this work: Firstly, the BβArg448Lys fibrinogen variant affects clot structural and functional properties in T2DM patients. Secondly, α2-AP is the protein responsible for the differences in lysis of recombinant BβArg448Lys fibrinogen variants in the plasma environment. And lastly, I proposed the AαTrp334Cys/Asn335Tyr changes in the Birmingham II patient are responsible for her clinical presentation. Accordingly, the aims of this work were to identify any additional effect of BβArg448Lys on fibrin network in type 2 diabetes, study the mechanisms behind the effects of the BβArg448Lys mutation on fibrin clot structural and functional properties, and finally, investigate the effect of fibrinogen AαTrp334Cys/Asn335Tyr on clot structure and lysis and abnormal patient phenotype. Compact clots with resistance to fibrinolysis were detected in carriers of Bβ448Lys variant of fibrinogen with diabetes. A direct role of the polymorphism in the changes observed with plasma clots was identified, and this may add to an already increased level of vascular risk in women with diabetes. In a recombinant system, differences in lysis between BβArg448Lys variants were attributable to the interplay between porosity of the clot, the fibrin-α2-AP interaction and plasmin generation. Finally, we confirm the AαTrp334Cys/Asn335Tryr substitutions in the fibrinogen BII patient are responsible for a clinical presentation of bleeding and thrombosis, providing mechanistic explanations for this patient phenotype. In summary, I extensively investigated the interplay between the fibrinogen molecule, fibrin clot structure, fibrinolysis and the environment and made a number of novel observations. Data generated has shed light on the mechanisms of increased thrombosis risk in patients with compact clots made of thin fibres, through genetic or environmental influence, which in turn will help to develop effective treatment strategies to reduce ischemic events in high risk patients.

No description available.

Exposure to acute bouts of whole body vibration (WBV), which can be employed as a novel form of exercise, has been reported to increase local skeletal muscle blood flow. However, the mechanism for this effect remains unclear. Therefore, this research aimed to explore the mechanism that would explain the effect of vibration on the peripheral cardiovascular function. Initially, the aim was to investigate the potential mechanism of the effect of WBV on the systemic blood flow, since there are currently no studies reporting any systemic effects of WBV on blood flow. The results did not demonstrate any systemic effects on blood flow (i.e. forearm blood flow) in response to acute unloaded and loaded squats with WBV. It was concluded that it was difficult to identify the effects of vibration on systemic cardiovascular function because, most likely due to the higher exercise intensity, skeletal muscle activation resulted in a decrement in blood flow from a distal site (i.e. forearm) to the main site (i.e. lower limb). Through the development of experimental methods involving applying vibration passively to the lower limbs, which avoids any influence of direct skeletal muscle activation and focuses solely on the mechanism inducing effects, it was demonstrated that ankle systolic blood pressure and ankle brachial pressure index substantially decreased in the post-vibration period. It was concluded that vibration has a direct effect on the peripheral cardiovascular function via increased vasodilatation; however, the mechanism underlying this effect remained unresolved. The effects of different durations of passive vibration on the peripheral circulation were also investigated and the results demonstrated that a longer duration of passive vibration (i.e. 8 minutes) resulted in a significantly higher lower leg blood flow during the recovery period than a shorter duration (i.e. 1, 2 and 4 minutes) of passive vibration. These data provide evidence for a greater effect of WBV occurring with a longer duration on the peripheral cardiovascular function, caused by the vasodilatation response throughout the recovery period. However, there might be a minimum effect of skeletal muscle activation occurring with a longer duration of passive vibration that leads to a direct response to localised heating. Furthermore, the thesis attempted to distinguish the effects of passive vibration on skeletal muscle activation from those on the peripheral vascular system. An experiment was designed in which passive vibration was applied with and without circulatory occlusion, to examine whether there was any underlying skeletal muscle activation. It was found that vibration with intact circulation produces more heat than the control, no vibration and occlusion, and occlusion plus vibration conditions. These effects were reflected by the higher skin temperature observed during exposure to vibration, and continuing into recovery. These data provide evidence that passive vibration does not appear to induce an increase in muscle activity. The data also suggest that the mechanism of the rise in skin temperature in response to passive vibration exposure is due to a vasodilatation that occurred in the lower limb via inducing an increase in shear stress at the blood vessels wall and led to an increase in circulating blood flow during exposure that continues into recovery. Overall, the results obtained demonstrate that vasodilatation occurs during and after vibration exposure and appears to be a process that is independent of skeletal muscle activation. It is postulated that the stimulus is a direct effect on the blood vessels via inducing an increase in shear stress that results in an increased vasodilatation, thereby increasing blood flow. Hence, these observations demonstrate that vibration stimulus has a direct effect on the muscle vascular bed as a primary effect and that there is no carry over effect into the systemic circulation. Thus, the results of this thesis indicate that vibration induced enhancement in the peripheral circulation could be using as a training stimulus and also could have a beneficial effect in assisting recovery routines from exertion.

Vascular smooth muscle cell (VSMC) phenotypic switching, from a contractile to a migratory phenotype, is essential for vascular repair and is compromised in ageing. Phenotypic transition involves dramatic actin reorganisation which is regulated by the linker of the nucleoskeleton and cytoskeleton (LINC) complex that spans the nuclear envelope (NE). The LINC complex physically anchors cytoskeletal filaments to the nucleoplasm via nesprin-SUN-nuclear lamina connections which enable rapid biophysical signalling between the nuclear interior and exterior. The nuclear lamina consists of A and B-type lamins that maintain nuclear architecture, however, the lamin A precursor protein, prelamin A, is a biomarker of VSMC ageing and this project investigates the impact of prelamin A upon LINC complex function. During VSMC ageing in-vitro, prelamin A accumulates at the presenescent growth phase which is associated with cellular elongation and focal adhesion reorganisation. Interference reflection imaging (IRM) and time-lapse microscopy revealed that presenescent VSMCs exhibit increased focal adhesion turnover with enhanced migratory speed and persistence. Importantly, prelamin A accumulation induced by siRNA-mediated knockdown of its processing enzyme, FACE-1, reiterates these morphological changes and enhances migratory persistence. Moreover, RhoA and Rac1 are well-established regulators of cell motility and their expression and activity diminishes in presenescent and FACE-1 depleted VSMCs. Fluorescence recovery after photobleaching (FRAP) also revealed that nuclear lamina disruption increases nesprin-2 dynamics at the nuclear exterior. Thus, we suggest that prelamin A impacts on actin-regulated processes including cell shape and motility via the LINC complex. We utilised an siRNA-mediated approach to investigate the importance of other LINC complex components in regulating cell morphology and migration. Interestingly, SUN2 levels decrease during in-vitro VSMC ageing and SUN2 knockdown enhances the migratory speed of VSMCs and fibroblasts similarly to presenescent VSMCs. The role of nesprins in regulating cell phenotype varies between different cells, highlighting that LINC complex organisation and function is flexible and cell-type specific. Together, our data reveal that the LINC complex is a versatile structure that is specialised to cell function and is an important regulator of cellular morphology, focal adhesion organisation, Rho GTPase activity and migration. VSMC ageing is associated with prelamin A accumulation and loss of SUN2 expression which consequently deregulates LINC complex organisation and functioning. Therefore, LINC complex disruption gives rise to an aged VSMC phenotype which we predict may underlie cardiovascular diseases such as atherosclerosis. In addition, IRM captured the release of adhesion-like structures, termed cell traces, from the rear of migrating VSMCs that outline their migratory path. Cell traces form physical tracks that enhance the speed and migrational directionality of neighbouring VSMCs. Therefore, we predict that cell traces support VSMC migration to injury sites and are important for vessel repair.

No description available.

No description available.