The receptor tyrosine kinase Tie-1 is a novel endothelial cell membrane receptor, with still unknown ligand, which seems to play an important role in vessel maturation. In our study, we show that VEGF regulates the proteolytic cleavage of the Tie-1 extracellular domain resulting to the release of an extracellular fragment and the production of an intracellular fragment with potential downstream signalling functions. This is mediated by a metalloprotease, involving phosphorylation of tyrosine kinases. Nitric oxide, thrombin, angioprotein II, Bradykinin and bFGF were not found to regulate proteolytic cleavage of the Tie-1 receptor's extracellular domain. Cell survival and cell proliferation experiments confirmed that the Tie-1 ectodomain cleavage leads to enhanced endothelial cell survival and proliferation. In order to examine the physiological role of Tie-1 cleavage we attempted to specifically inhibit truncation by synthesising a peptide corresponding to the truncation site and raising a monoclonal antibody against it. The in-vivo angiogenesis model of the chick chorioallantoic membrane (CAM) model was used. Tie-1 was identified in the developing CAM existing in both its truncated and full-length form. Treatment of the mature CAM with VEGF results to ectodomain cleavage of Tie-1. treatment of the CAM with monoclonal antibodies recognising the extracellular epitope of the receptor results to interstitial oedema suggesting a role for the receptor in vessel stabilisation. Tie-1 was also identified in platelets where it is present in a different, non-cleavable isoform suggesting that proteolytic cleavage of the Tie-1 receptor's extracellular domain is endothelial cell specific phenomenon.

Carbon monoxide (CO) is an important signalling messenger in mammalian cells as it participates in a variety of physiological processes including vessel tone regulation. Dr. Motterlini's group has discovered a new class of molecules which have the ability to carry and deliver CO to physiological systems. These molecules were termed CO-releasing molecules (CO-RMs) and they are an excellent tool to investigate the biological role of CO in the vasculature and other systems. The major focus of the research presented in this report was to evaluate the effects of different water soluble CO-RMs on vascular tone. For this purpose, an aortic ring preparation model was used to assess the vasodilatory properties of CORM-3 and CORM-A1, the first two water-soluble CO-RMs to be identified, and the cellular targets involved in this effect. CORM-3 is a transition metal carbonyl that liberates CO very rapidly (1=1-5 min) in physiological solutions, whereas CORM-A1 is a boron-containing carbonate with a much slower rate of CO release (tvi=21 min at pH=7.4). In the current studies CORM-3 induced a rapid endothelium-dependent vasorelaxation, whereas CORM-A1 elicited gradual endothelium-independent vasorelaxation. The inactive form of both CO-RMs, in which CO has been deliberately depleted, did not exert vasorelaxation indicating a direct involvement of CO liberated from the compounds in the observed vasorelaxation. The vasorelaxation induced by both molecules was enhanced and attenuated by an activator and inhibitor of guanylate cyclase (sGC), respectively. CORM-3-mediated vasorelaxation was completely abolished by non-selective inhibitors of potassium channels (K+), and partially attenuated by inhibition of ATP dependant (Katp) potassium channels. In contrast, CORM-A1 -mediated vasorelaxation was partially attenuated by non selective inhibition of K+ and by inhibition of voltage dependent (Ky) potassium channels. Even at concentrations higher than that used to induce significant vasorelaxation, both CO-RMs had no noticeable effect on the viability of rat aortic smooth muscle cells (A7r5) in vitro. CORM-319, a new water soluble iron containing CO-RM, also induced significant vasorelaxation and was relatively safe to cultured SMCs compared to other non-water soluble iron containing CO-RMs that were extremely toxic. In summary, our data reveal that the CO-RMs examined in this project are promising CO carriers that could be further modified for optimal therapeutic applications. In addition, our data demonstrate the significant effect imposed by the chemical structure and kinetics of CO release on the pharmacological activity of various CO-RMs.

The regulation of vascularisation is a major challenge facing osteochondral tissue engineering. Articular cartilage is an avascular tissue and the presence of blood vessels is associated with various pathologies of the tissue. Conversely, bone requires an extensive vascular supply in order to maintain viability and function. Angiogenesis is influenced by aspects of the microenvironment including extracellular matrix molecules and cell secreted factors. In this thesis, in vitro models were used to investigate the effects of glycosaminoglycan content and bone marrow-derived mesenchymal stromal cell secretome on angiogenesis in context to osteochondral tissue. In addition, an in vivo study was used to determine the effect of endocultivation time pre-BMP-2 application on the vascularisation of bone tissue engineered by endocultivation. This thesis has demonstrated that articular cartilage glycosaminoglycans inhibit the adhesion of endothelial cells and suggests this activity is due to chondroitin sulphate and/or hyaluronan. Furthermore, this thesis has also shown that when chondrogenically or osteogenically differentiated, bone marrow-derived mesenchymal stromal cells reduce production of angiogenesis-related proteins and display anti-angiogenic properties in vitro. The results presented in this thesis support the use of endocultivation as a technique for tissue engineering vascularised bone grafts in vivo, however, suggest that increased vascular ingrowth may not necessarily lead to increased bone formation. Together, these findings demonstrate how the cell microenvironment may influence the vascularisation of osteochondral tissue which may have important implications for osteochondral tissue engineering and provide insight into how pathological vascularisation of articular cartilage may occur.

Endothelial cells (ECs) form the innermost layer of all blood vessels, and their function and behaviour are important in angiogenesis. VEGF is the most potent angiogenic factor stimulating EC proliferation, migration, and differentiation as well as survival. Vascular endothelial growth factor receptor 2 (VEGFR2) is the major VEGF receptor which mediates VEGF-induced angiogenesis on ECs. Neuropilin 1 (NRPI) is a VEGF co-receptor that enhances VEGF-stimulated VEGFR2 angiogenic signals. A disintegrin and metalloproteinase with thrombospondin repeats (ADAMTS)-I, -2, -5, -8, and -9 have been shown to be anti-angiogenic. Our preliminary data suggested that ADAMTS-4 was expressed by two types of ECs, human umbilical vein endothelial cells (HUVECs) and human dennal microvascular endothelial cells (HuDMECs), and it inhibited endothelial cell differentiation so this thesis tested the hypothesis that ADAMTS-4 may be an anti-angiogenic molecule. For the first time, this thesis demonstrated that ADAMTS-I bound to NRPI by co-IP, and that ADAMTS-4 may also interact with NRPI. These potential bindings between ADAMTSs and NRP 1 may play roles in neuron system such as neuron development and axon guidance or in angiogenesis. These new interactions between ADAMTS-I, ADAMTS-4 and VEGF signalling complexes may point to new mechanisms of angiogenesis as NRPI has been reported to be more than just a VEGF co-receptor. Also, using co-IP an interaction between ADAMTS-4 and VEGF was demonstrated for the first time which may result in its effects on VEGF-mediated HuDMEC behaviour. Indeed, ADAMTS-4 reduced VEGF-stimulated VEGFR2 phosphorylation and also inhibited VEGF-induced HuDMEC differentiation and migration suggesting that ADAMTS-4 has anti-angiogenic activity. Sequence alignment of all 19 human ADAMTSs showed a high degree of similarity in the thrombospondin repeats suggesting that all 19 ADAMTSs could be anti-angiogenic.

3-D visualization of blood vessel from standard medical datasets (e.g. CT or MRI) play an important role in many clinical situations, including the diagnosis of vessel stenosis, virtual angioscopy, vascular surgery planning and computer aided vascular surgery. However, unlike other human organs, the vasculature system is a very complex network of vessel, which makes it a very challenging task to perform its 3-D visualization. Conventional techniques of medical volume data visualization are in general not well-suited for the above-mentioned tasks. This problem can be solved by reconstructing vascular geometry. Although various methods have been proposed for reconstructing vascular structures, most of these approaches are model-based, and are usually too ideal to correctly represent the actual variation presented by the cross-sections of a vascular structure. In addition, the underlying shape is usually expressed as polygonal meshes or in parametric forms, which is very inconvenient for implementing ramification of branching. As a result, the reconstructed geometries are not suitable for computer aided diagnosis and computer guided minimally invasive vascular surgery. In this research, we develop a set of techniques associated with the geometry reconstruction of vasculatures, including segmentation, modelling, reconstruction, exploration and rendering of vascular structures. The reconstructed geometry can not only help to greatly enhance the visual quality of 3-D vascular structures, but also provide an actual geometric representation of vasculatures, which can provide various benefits. The key findings of this research are as follows: 1. A localized hybrid level-set method of segmentation has been developed to extract the vascular structures from 3-D medical datasets. 2. A skeleton-based implicit modelling technique has been proposed and applied to the reconstruction of vasculatures, which can achieve an accurate geometric reconstruction of the vascular structures as implicit surfaces in an analytical form. 3. An accelerating technique using modern GPU (Graphics Processing Unit) is devised and applied to rendering the implicitly represented vasculatures. 4. The implicitly modelled vasculature is investigated for the application of virtual angioscopy.

In recent years it has become apparent that hydrogen sulphide (H2S) is an important biological mediator. In the vasculature, it produces complex responses: contraction in some blood vessels, relaxation in others, via multiple mechanisms. This thesis examined the relationship between H2S and oxygen in determining vascular responsiveness, and was conducted using porcine splenic and mesenteric arteries. Studies were also conducted using porcine splenic veins, since few studies have examined venous function. Additionally, studies were extended to the resistance vasculature by determining responses to a H2S donor in small arteries isolated from the rat mesentery. Porcine vessels were set up in an isometric tension recording system and rat small mesenteric arteries were set up in a pressure myograph. Vessels were pre-contracted and responses to the H2S donor, NaHS, were generated in the presence and absence of putative inhibitors, under either 95% O2:5% CO2, 95% air:5% CO2 or 95% N2:5% CO2 gassing conditions. Generally, in both porcine arteries and veins, when gassing with higher oxygen levels (95% O2:5% CO2 or 95% Air:5% CO2), NaHS induced contractile responses, whereas gassing with a lower oxygen level (95% N2:5% CO2), NaHS induced vasorelaxation. At higher O2 levels, removal of the endothelium or, the nitric oxide (NO) synthase inhibitor L-NAME, significantly attenuated contractile response in all porcine vessels. This suggests an interaction between endothelium-derived NO and NaHS, whereby the removal of the vasorelaxatory influence of NO resulted in contraction. In porcine arteries, relaxation at lower O2 levels was attenuated by glibenclamide, suggesting that NaHS activated KATP channels to cause relaxation. In porcine veins, removal of the endothelium or, L-NAME, abolished NaHS-induced relaxation, showing this relaxation occurred via the release of endothelium-derived NO. In rat mesenteric small arteries responses to NaHS did not change with different O2 levels and NaHS-induced vasodilatation that was abolished by desensitization of sensory nerves with capsaicin or the presence of BIBN 4096. These observations suggest NaHS-induced vasodilatation is mediated via release of CGRP from sensory nerves. Thus, responses to NaHS in large conduit arteries and veins, are sensitive to the prevailing level of O2 the tissue is exposed to. At more physiological levels of O2 the predominant response is a vasorelaxation, mediated by either, activation of KATP channels in arteries or, the release of NO in veins. In small arteries, the predominant response is a vasodilator response, involving the release of neuropeptides from sensory nerves. The predominance of a vasorelaxant/vasodilator response is consistent with the observation that mice which lack the capacity to generate endogenous H2S are hypertensive.

Previously, acyl derivatives of CoA were shown to antagonise human native and recombinant P2Y1 purine receptors. The main aim of this thesis was to study the effect of these endogenous nucleotide derivatives at endogenous P2Y1 receptors in blood vessels. Using isometric tension recordings, CoA, acetyl-CoA and palmitoyl-CoA (PaCoA) appeared to show selectivity for P2Y1 receptors (over P2Y2 and adenosine receptors) in the rat isolated thoracic aorta, with PaCoA being the most potent of the CoA derivatives used. In porcine isolated mesenteric arteries (PMA) and porcine isolated coronary arteries (PCA), isometric tension recordings indicated that ADP mediated endothelium-dependent and endothelium-independent relaxations, respectively. Relaxations in PMA were blocked by the P2Y1 receptor antagonist MRS2500 and PaCoA, whilst these were ineffective against ADP relaxations in the PCA. A FlexStation was used to monitor calcium responses in native HEK293 cells expressing P2Y1 and P2Y2 receptors using ADP and UTP, respectively. Responses to UTP were not significantly altered in the presence of PaCoA. In contrast, ADP-evoked responses were significantly inhibited in the presence of either MRS2500 or PaCoA. These data raise the possibility of an endogenous selective antagonism of P2Y1 receptors via CoA compounds, irrespective of species or cellular environment. Nicotinamide adenine dinucleotide (NAD) is an intracellular nucleotide which has been identified as an agonist at P2Y1, P2Y11, P2X and adenosine receptors. NAD evoked endothelium-independent concentration-dependent contractions of the pre-contracted PMA, which were unaltered in the presence of PaCoA. In contrast, αβ-methylene ATP (a desensitizing P2X receptor agonist) significantly reduced these responses suggesting the involvement of P2X1-like receptors. In both RTA and PCA, NAD evoked endothelium-independent concentration-dependent relaxations of the pre-contracted vessels, which were attenuated by SCH58261, but not PaCoA, which suggests the involvement of smooth muscle A2A receptors. These results together emphasise the possibility of a tissue and receptor-specific role of NAD as an endogenous extracellular nucleotide in purinergic signalling.