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International audience; Connexins are proteins forming gap junction channels for intercellular communication. Connexin40 (Cx40) is highly expressed by endothelial cells (ECs) of healthy arteries but this expression is lost in ECs overlying atherosclerotic plaques. Low/oscillatory shear stress observed in bends and bifurcations of arteries is atherogenic partly through activation of the pro-inflammatory NFκB pathway in ECs. In this study, we investigated the relation between shear stress, Cx40 and NFκB. Shear stress-modifying casts were placed around carotid arteries of mice expressing eGFP under the Cx40 promoter (Cx40+/eGFP ). We found that Cx40 expression is decreased in carotid regions of oscillatory shear stress but conserved in high and low laminar shear stress regions. These results were confirmed in vitro. Using phage display, we retrieved a binding motif for the intracellular regulatory Cx40 C-terminus (Cx40CT), i.e. HS[I, L, V][K, R]. One of the retrieved peptides (HSLRPEWRMPGP) showed a 58.3% homology with amino acids 5-to-16 of IκBα, a member of the protein complex inhibiting NFκB activation. Binding of IκBα (peptide) and Cx40 was confirmed by crosslinking and en face proximity ligation assay on carotid arteries. TNFα-induced nuclear translocation of NFκB in ECs was enhanced after reducing Cx40 with siRNA. Transfection of HeLa cells with either full-length Cx40 or Cx40CT demonstrated that Cx40CT was sufficient for inhibition of TNFα-induced NFκB phosphorylation. Finally, Tie2CreTgCx40fl/flApoe-/- mice showed exaggerated shear stress-induced atherosclerosis and enhanced NFκB nuclear translocation. Our data show a novel functional IκBα-Cx40 interaction that may be relevant for the control of NFκB activation by shear stress in atherogenesis.

Supplemental Digital Content is available in the text., Background: Myeloid cells are central to atherosclerotic lesion development and vulnerable plaque formation. Impaired ability of arterial phagocytes to uptake apoptotic cells (efferocytosis) promotes lesion growth and establishment of a necrotic core. The transcription factor interferon regulatory factor (IRF)-5 is an important modulator of myeloid function and programming. We sought to investigate whether IRF5 affects the formation and phenotype of atherosclerotic lesions. Methods: We investigated the role of IRF5 in atherosclerosis in 2 complementary models. First, atherosclerotic lesion development in hyperlipidemic apolipoprotein E-deficient (ApoE-/-) mice and ApoE-/- mice with a genetic deletion of IRF5 (ApoE-/-Irf5-/-) was compared and then lesion development was assessed in a model of shear stress-modulated vulnerable plaque formation. Results: Both lesion and necrotic core size were significantly reduced in ApoE-/-Irf5-/- mice compared with IRF5-competent ApoE-/- mice. Necrotic core size was also reduced in the model of shear stress-modulated vulnerable plaque formation. A significant loss of CD11c+ macrophages was evident in ApoE-/-Irf5-/- mice in the aorta, draining lymph nodes, and bone marrow cell cultures, indicating that IRF5 maintains CD11c+ macrophages in atherosclerosis. Moreover, we revealed that the CD11c gene is a direct target of IRF5 in macrophages. In the absence of IRF5, CD11c- macrophages displayed a significant increase in expression of the efferocytosis-regulating integrin-β3 and its ligand milk fat globule-epidermal growth factor 8 protein and enhanced efferocytosis in vitro and in situ. Conclusions: IRF5 is detrimental in atherosclerosis by promoting the maintenance of proinflammatory CD11c+ macrophages within lesions and controlling the expansion of the necrotic core by impairing efferocytosis.

Evidence is accumulating that calcium-rich microdeposits in the vascular wall might play a crucial role in the onset and progression of atherosclerosis. Here we investigated an atherosclerotic lesion of the carotid artery in an established murine model, i.e. the apolipoprotein E-deficient (APOE(-/-) ) mouse to identify (i) the presence of microcalcifications, if any, (ii) the elemental composition of microcalcifications with special reference to calcium/phosphorus mass ratio and (iii) co-localization of increased concentrations of iron and zinc with microcalcifications. Atherosclerosis was induced by a flow-divider placed around the carotid artery resulting in low and high shear-stress regions. Element composition was assessed with a proton microprobe. Microcalcifications, predominantly present in the thickened intima of the low shear-stress region, were surrounded by areas with normal calcium levels, indicating that calcium-precipitation is a local event. The diameter of intimal microcalcifications varied from 6 to 70 μm. Calcium/phosphorus ratios of microcalcifications varied from 0.3 to 4.8, mainly corresponding to the ratio of amorphous calcium-phosphate. Increased iron and zinc concentrations commonly co-localized with microcalcifications. Our findings indicate that the atherosclerotic process in the murine carotid artery is associated with locally accumulated calcium, iron and zinc. The calcium-rich deposits resemble amorphous calcium phosphate rather than pure hydroxyapatite. We propose that the APOE(-/-) mouse, in which atherosclerosis was evoked by a flow-divider, offers a useful model to investigate the pathophysiological significance of accumulation of elements such as calcium, iron and zinc.

Coronary atherosclerosis is a major cause of mortality and morbidity worldwide. Despite its systemic nature, atherosclerotic plaques form and develop at “predilection” sites often associated with disturbed biomechanical forces. Therefore, computational approaches that analyse the biomechanics (blood flow and tissue mechanics) of atherosclerotic plaques have come to the forefront over the last 20 years. Assignment of appropriate material properties is an integral part of the simulation process. Current approaches for derivation of material properties rely on macro-mechanical testing and are agnostic to local variations of plaque stiffness to which collagen microstructure plays an important role. In this work we used Atomic Force Microscopy to measure the stiffness of healthy and atherosclerotic coronary arteries and we hypothesised that are those are contingent on the local microstructure. Given that the optimal method for studying mechanics of arterial tissue with this method has not been comprehensively established, an indentation protocol was firstly developed and optimised for frozen tissue sections as well as a co-registration framework with the local collagen microstructure utilising the same tissue section for mechanical testing and histological staining for collagen. Overall, the mechanical properties (Young’s Modulus) of the healthy vessel wall (median = 11.0 kPa, n=1379 force curves) were found to be significantly stiffer (p=1.3410-10) than plaque tissue (median=4.3 kPa, n=1898 force curves). Within plaques, lipid-rich areas (median=2.2 kPa, n=392 force curves) were found significantly softer (p=1.4710-4) than areas rich in collagen, such as the fibrous cap (median=4.9 kPa, n=1506 force curves). No statistical difference (p=0.89) was found between measurements in the middle of the fibrous cap (median=4.8 kPa, n=868 force curves) and the cap shoulder (median=5.1 kPa, n=638 force curves). Macro-mechanical testing methods dominate the entire landscape of material testing techniques. Plaques are very heterogenous in composition and macro-mechanical methods are agnostic to microscale variations in plaque stiffness. Mechanical testing by indentation may be better suited to quantify local variations in plaque stiffness, that are potent drivers of plaque rupture. Open Access

This thesis describes the design, synthesis, and testing of novel MRI contrast agents based on iron oxide nanoparticles for the specific detection and identification of vulnerable plaque. Cardiovascular disease remains the leading cause of death worldwide, primarily due to heart attacks and strokes resulting from vulnerable plaque rupture. Vulnerable plaque occurs in atherosclerosis, when plaque is deposited in the walls of blood vessels and can take on vulnerable or stable phenotypes. There is an urgent need for an imaging biomarker to enable the specific detection of vulnerable plaque to facilitate treatment and prevent future heart attacks and strokes. MRI is a non-ionising, non-invasive imaging modality with the potential for highly customisable contrast agents. Chapter 2 discusses the design and synthesis of a library of superparamagnetic iron oxide nanoparticle-based contrast agents through thermal decomposition and coating with different surface ligands, including poly (maleic anhydride-alt-1-octadecene), alendronate, and poly (ethyleneimine). The agents were characterised through transmission electron microscopy, dynamic light scattering, zeta potential and relaxivity measurements. Chapter 3 presents the results of biological tests performed using the lead candidate from the synthesis carried out in chapter 2, including the coupling of a targeting antibody to the contrast agent, in vitro testing, and MR imaging in a preclinical model with histological verification of the results. Chapter 4 builds on the initial design and synthetic work of Chapter 2 with the introduction of a gold shell, moving towards multi-modality imaging. Several synthetic routes for the introduction of the gold shell and a selection of surface ligands including poly (maleic anhydride-alt-1-octadecene), homocysteine, and citrate were studied, and characterised through transmission electron microscopy, dynamic light scattering, and zeta potential measurements. Chapter 5 summarises the conclusions of the project, presents potential areas for future work and concludes the thesis. Open Access

The core goal of synthetic biology as a discipline is to design, develop and characterize biological parts in order to precisely control cellular behaviour. Much of the research in this field has been focused on the development of gene regulatory networks, namely switches and oscillators. The study of synthetic gene oscillators has attracted significant attention in the past decade due to their intriguing dynamics and relevance in controlling inflammatory, metabolic and circadian signalling pathways. Additionally, the precise expression dynamics and molecular mechanisms that underlie the mammalian circadian clock structure are not fully understood. The work presented herein regards the design and implementation of a tuneable mammalian synthetic gene oscillator with a novel biological structure. To this end, an approach based on a combination of in silico design and in vivo part validation, in conjunction with a comparative analysis of previously implemented synthetic gene oscillators, was taken when assembling the proposed system. The topology of the system relies on a delayed negative feedback loop, consisting of the coupled regulatory activities of the transcription regulators LacI, tTA, and Gal4. The numerical solution and stability analysis of an ODE-based model describing the dynamics of the system are indicative that the proposed system is capable of generating sustained oscillations across a wide range of parameter values. The biological parts that comprise the system have been monitored and validated in HEK293T cells through time-lapse fluorescence microscopy and image analysis. The in vivo performance of the proposed mammalian synthetic gene oscillator was also assessed in the HEK293T cell line, and monitored using time-lapse fluorescence microscopy. Damped fluorescence oscillations were observed: these could be tuned by a differential IPTG concentration gradient and abolished by doxycycline. The proposed mammalian synthetic gene oscillator provides valuable insight into the gene expression regulatory processes leading to oscillatory behaviour, and has the potential to foster progress in future synthetic biology-based therapies. Open Access

As the physical boundary separating the cell interior from the outside world, the plasma membrane is arguably one of the most important cellular organelles. Along with the proteins embedded within it, the plasma membrane plays a part in a number of vital roles including intercellular communication, cell motility and homeostasis. Despite this, comparatively little is known about the exact structure of the plasma membrane. The plasma membrane is a lipid bilayer that is widely believed to undergo transient phase separation into domains of different viscosities, which may be responsible for certain membrane properties. By using molecular rotors (organic fluorophores whose fluorescence properties depend on the viscosity of their surrounding environment) in conjunction with fluorescence lifetime imaging microscopy (FLIM), it is possible to generate a viscosity map of a heterogeneous biological system. In this thesis, we use molecular rotors to investigate the viscoelastic properties and phase behaviour of a range of lipid bilayer systems. We begin by using molecular rotors based on a boron-dipyrrin (BODIPY) core to investigate membrane viscosity within model systems, finding that different lipid phases display different viscosities, and visualising lipid phase separation within giant vesicles. We go on to use BODIPY rotors to image viscosity within the plasma membranes of live Escherichia coli bacteria at different temperatures, finding that they display a much higher degree of membrane ordering than previously observed in eukaryotic cells. Next we explore synthetic alternatives to the conventionally used BODIPY rotors in order to address their shortcomings, before using molecular rotors based on a thiophene moiety to determine the viscosity of the plasma membranes of live human cell lines. Our results from these studies suggest that phase-separation may indeed occur within the plasma membrane. Open Access

The prevalent pathology leading to high mortality from cardiovascular disease is atherosclerosis. Despite strong evidence for the association of shear stress and the site-specificity of atherosclerosis initiation, there is a lack of treatment focusing on modulating shear stress-induced phenotypes in endothelial cells. The overarching theme of the thesis is to uncover the causal molecular networks that are activated by mechanical forces in endothelial cells. We present an integrated platform for gene network inference and validation, which integrates network inference from gene expression data with functional genomics within a single framework to set up an iterative cycle of measurement and network refinement. In order to achieve this, a number of technological innovations were necessary and developed. We present an approach, named ARNI, to logically model and construct through abductive reasoning, regulatory gene networks from gene expression profiles and background prior knowledge on gene functions and interactions. We demonstrate the improved predictive power and complexity of our inferred network topologies compared with those generated by other non-symbolic inference approaches. A bioinformatics workflow for the integration of gene expression profiles across platforms and species is presented and applied for generating an integrated endothelial cell mechanoresponsive gene expression profile. The integrated dataset identified >1600 genes to be shear responsive, more than any other study, and in this gene set all known mechanosensitive genes and pathways were present. We also present the first high-throughput RNAi platform to demonstrate successful reverse electroporation of mammalian primacy cell lines and which couples RNAi screening with mechanotransduction studies. Each step of the assemblage is evaluated and effective loss-of-function is demonstrated for multiple gene targets. The tools presented in this thesis provide the basis for developing qualitative models and targeted therapies, in order to further our pursuit for shear-mediated atherosclerosis prevention. Open Access

Background— Although disturbed flow is thought to play a central role in the development of advanced coronary atherosclerotic plaques, no causal relationship has been established. We evaluated whether inducing disturbed flow would cause the development of advanced coronary plaques, including thin cap fibroatheroma. Methods and Results— D374Y -PCSK9 hypercholesterolemic minipigs (n=5) were instrumented with an intracoronary shear-modifying stent (SMS). Frequency-domain optical coherence tomography was obtained at baseline, immediately poststent, 19 weeks, and 34 weeks, and used to compute shear stress metrics of disturbed flow. At 34 weeks, plaque type was assessed within serially collected histological sections and coregistered to the distribution of each shear metric. The SMS caused a flow-limiting stenosis, and blood flow exiting the SMS caused regions of increased shear stress on the outer curvature and large regions of low and multidirectional shear stress on the inner curvature of the vessel. As a result, plaque burden was ≈3-fold higher downstream of the SMS than both upstream of the SMS and in the control artery ( P P P P Conclusions— These data support a causal role for lowered and multidirectional shear stress in the initiation of advanced coronary atherosclerotic plaques. Persistently lowered shear stress appears to be the principal flow disturbance needed for the formation of thin cap fibroatheroma.

Atherosclerosis is a focal inflammatory disease, non-uniformly distributed in regions of disturbed flow within the arterial system. The local accumulation of lipids and inflammatory cells in these regions drives lesion initiation and progression into advanced plaques. Along with systemic risk factors, local haemodynamic metrics, such as wall shear stress (WSS), have been shown to be good correlates to plaque development. Few studies have investigated the relationship between mechanics and biology longitudinally over plaque development. However, conflicting views on the role of low and high wall shear stress in the development of the disease remain. Using high-resolution imaging and novel techniques like 3-D histology, we seek to provide more conclusive evidence of the correlation between perturbed haemodynamics and markers of atherosclerosis in two longitudinal studies in different animal models. The first study comprised of the development of a computational workflow to analyse co-localisations of selected disease markers with perturbed shear regions, induced in carotid arteries of ApoE-/- mice. Serial, in vivo mouse-specific haemodynamics were computed in micro-CT based geometries and coupled with plaque distribution spatially mapped on the in vivo lumen at each time point. Two novel haemodynamic metrics and other established metrics were assessed to find the best predictor of local areas of plaque formation. A quantitative analysis revealed plaque lipids and macrophages in the initial stage of the disease best overlapped with our custom metric, the low shear index (LSI), suggesting the importance of low shear in the initiation of atherosclerosis. In the second study, the developed workflow was applied to correlate plaque biology and mechanical metrics in transgenic hypercholesterolemic Yucatan minipigs. Lesions were induced by implanting a stenotic stent in one of the coronaries, and in vivo haemodynamics were computed in OCT based coronary reconstructions serially from healthy to advanced disease stages. Animals developed advanced human-like atherosclerotic lesions in regions of sustained low and oscillatory shear, induced by the stent. Histology based macrophage distribution showed good correlations with the newly developed metric, LSI, further emphasising the importance of low shear in atherogenesis. In conclusion, we have developed an integrated platform for high-resolution studies of plaque mechanobiology. Low shear was the best correlate to markers of atherosclerosis in two animal models, quantified by our novel metric – LSI, which might be a good predictor of disease development. Additionally, this thesis shows the importance of longitudinal in vivo studies of atherosclerosis and contributes to further understanding of this disease, while also providing tools that can be further extended in future studies. Open Access

Rupture of “vulnerable” atherosclerotic plaques and subsequent thrombosis cause acute cardiovascular events, and can develop upon exposure of the arterial wall to low shear stress. Myeloid cells - the main inflammatory cells within atherosclerotic plaques - are heterogeneous; ranging from “classical” pro-inflammatory M1 macrophages to “alternative” M2 macrophages and various subsets of dendritic cells. The activation of Toll-like receptors and downstream Interferon Regulatory Factors (IRFs) is involved in atherosclerosis. IRF5 polarises macrophages towards the M1 phenotype and modulates cytokine production by dendritic cells. I utilised two murine models of atherosclerosis: the hypercholesterolaemic ApoE-/- (Apolipoprotein E knockout) mouse strain, and a perivascular cast modifying shear stress patterns in the carotid artery. Firstly, I found the majority of macrophages in early and intermediate lesions of the aortic root and advanced oscillatory shear stress-modulated lesions express heme oxygenase-1 (HO-1). The representation of the M1 macrophage marker iNOS (inducible nitric oxide synthase) and IRF5 is more prevalent in low shear stress-modulated plaques, which resemble a vulnerable plaque, while M2 macrophage markers are elevated in oscillatory shear stress-modulated plaques resembling stable plaques. Secondly, I studied the effect of IRF5 deletion on the development of atherosclerosis by comparing the severity of atherosclerosis in ApoE-/- mice with ApoE-/-IRF5-/- mice. Atherosclerotic lesions in the aortic root of ApoE-/-IRF5-/- mice are reduced in size, and in all vascular regions they have smaller necrotic cores (a marker of plaque vulnerability), due to a reduction in efferocytosis, and an increase in atheroprotective macrophages. Lesions in ApoE-/-IRF5-/- mice also have a depleted content of cells expressing CD11c; therefore IRF5 is detrimental in atherosclerosis by skewing myeloid cell differentiation towards dendritic cells possibly via GM-CSF. My study provides a novel link between inflammatory signalling, efferocytosis and necrotic core formation. Open Access

Atherosclerosis is the leading cause of cardiovascular disease, topping the mortality list in the United Kingdom and the rest of the world. Macrophage activation and polarisation are key steps in host defence and chronic inflammatory diseases, including atherosclerosis. The myeloid glycoprotein receptor CD200R1 belongs to a family of four isoforms and signals after binding to its cognate ligand, CD200. The CD200/CD200R1 interaction blocks pro-inflammatory cytokines and has never before been studied in atherosclerosis. My work has demonstrated that CD200R is weakly expressed in atherosclerotic lesions during disease progression and significantly down-regulated in secondary lymphoid organs. Moreover, changes in shear stress had no effect on the expression of CD200R in plaques. During the steady state, the expression of CD200R on circulating monocytes was highest on the CD11b+CD115+Ly6Cint subset, revealing a potential mechanism for regulation of maturing monocytes. In vitro studies demonstrated that CD200R expression is sensitive to M1 macrophage polarising cytokine IFN-γ and MyD88-dependent TLR ligands. In contrast, non-MyD88-dependent TLR3 signalling had no effect on its expression, supporting previous findings in the literature. Moreover, CD200R1 mRNA expression was induced on alternatively activated M2a macrophage subsets following IL-4 and IL-13 cytokine treatment, whereas oxLDL had no effect. Immunotherapy with CD200-Fc in ApoE-/- mice exerted no significant changes on lesion size and phenotype compared to controls after nine weeks. My findings indicate that the type of inflammatory stimulus may play a role in dictating the ability of myeloid cells to terminate their own activation via CD200/CD200R1 signalling. Hence, chronic inflammation may be promoted by reduced presence of inhibitory signals leading to sustained, unresolved inflammation. In summary, my work has revealed new insight into the regulation of the inhibitory CD200R pathway in atherosclerosis, the molecular signals that may affect the regulation of inflammation through CD200R and possible future therapeutic strategies. Open Access

Atherosclerosis is defined as the thickening of an artery due to the accumulation of fatty acids, cholesterol, and cellular debris within the blood vessel lining. This research aims to understand the influence of atherosclerotic plaque on residual stress within t he arterial layers. Three layers of human coronary artery were studied under the influence of blood loading. Residual stress due to the opening angle and longitudinal stretch was included in the numerical model of the artery. A new method is used for model ing the effect of the opening angle on the distribution of stress. The finite element method was used for the numerical modeling of the artery in both healthy and atherosclerotic conditions. Under the blood loading condition, it was found that the presence of plaque produces higher stress in the arterial wall. This study allows us to investigate how the presence of the plaque affects residual stress and the overall stress distribution in the artery wall. Open Access

Biological systems are usually complex nonlinear systems of which we only have a limited understanding. Here we show three different aspects of investigating such systems. We present a method to extract detailed knowledge from typical biological trajectory data, which have randomness as a main characteristic. The migration of immune cells, such as leukocytes, are a key example of our study. The application of our methodology leads to the discovery of novel random walk behaviour of leukocyte migration. Furthermore we use the gathered knowledge to construct the under- lying mathematical model that captures the behaviour of leukocytes, or more precisely macrophages and neutrophils, under acute injury. Any model of a biological system has little predictive power if it is not compared to collected data. We present a pipeline of how complex spatio- temporal trajectory data can be used to calibrate our model of leukocyte migration. The pipeline employs approximate methods in a Bayesian framework. Using the same approach we are able to learn additional information about the underlying signalling network, which is not directly apparent in the cell migration data. While these two methods can be seen as data processing and analysis, we show in the last part of this work how to assess the information content of experiments. The choice of an experiment with the highest information content out of a set of possible experiments leads us to the problem of optimal experimental design. We develop and implement an algorithm for simulation based Bayesian experimental design in order to learn parameters of a given model. We validate our algorithm with the help of toy examples and apply it to examples in immunology (Hes1 transcription regulation) and signal transduction (growth factor induced MAPK pathway).

Atherosclerosis is a chronic lipid-modulated inflammatory disease, which develops preferentially in arterial regions, where blood flow is disturbed. Shear stress is exerted on the endothelium and it has been shown that the tangential force can influence intracellular signalling cascades, modulating transcriptional activation or repression of mechanosensitive genes. The exact mechanisms of mechanotransduction are not entirely understood, but the shear-dependent transcription factor Kruppel-like factor 2 (KLF2) has been considered as the master switch in shear-controlled endothelial gene expression. Induction of its target genes triggers anti-inflammatory and anti-thrombotic signalling cascades, determining an atheroprotective phenotype. Endothelial KLF4 has also recently been shown to be shear-sensitive and that it can compensate for reduced KLF2 transcripts. Another member of the KLF family, KLF6, is known to be present in the endothelium, but has not been further investigated. This suggests that KLF2 might not be a single master regulator in the process of shear-dependent atheroprotection. Since KLFs have been demonstrated to form circuitries in other tissues, the hypothesis of the present study was that shear stress invokes a KLF2-KLF4-KLF6 network, which regulates the atheroprotective transcriptional program in the endothelium. The work aimed at understanding shear-dependent signalling mechanisms to potentially reveal therapeutic targets. Firstly, a flow device allowing the in vitro exposure of primary porcine aortic endothelial cells (PAECs) to shear stress was designed. Gene expression patterns were examined with the consideration of parameters associated with time, shear stress magnitude and interference with short inhibitory ribonucleic acid (siRNA). These studies were carried out using quantitative polymerase chain reaction (qPCR), genome-wide microarray analysis, protein detection and computational modelling. The findings revealed an oscillatory expression pattern for KLF2 and KLF4 in high shear regimes of 20 dyne/cm2. siRNA-mediated silencing of KLF4 resulted in a significant downregulation of KLF2 and this dependency was shown for the first time. Global genome analysis identified a number of novel shared, but also individual downstream targets, suggesting that the transcription factors have important non-compensatory regulatory functions. In conclusion, this work contributes to the understanding of the individual roles of KLF2 and KLF4 and their functional overlap in the mediation of atheroprotection. Further investigations are required to assess whether KLFs could potentially be useful in preventing atherosclerosis or identifying atherosclerosis treatments.

Atherosclerosis, a chronic inflammatory disease of arteries, occurs predominantly at regions of the arterial system that are exposed to disturbed patterns of blood flow. Blood flow influences the atherosclerosis by exerting shear stress on endothelial cells (ECs). Although the signalling pathways that activate pro-inflammatory NF-κB transcription factors are well defined, the regulation and physiological significance of differential NF-κB subunit expression is poorly understood. In this thesis, we demonstrate that RelA NF-κB sub-unit expression is positively regulated in ECs via c-Jun N-terminal kinase (JNK) and the transcription factor ATF2. This pathway promoted focal arterial inflammation as genetic deletion of JNK1 reduced RelA expression and macrophage accumulation at an athero-susceptible site. Furthermore, JNK signalling to RelA is controlled by mechanical forces as en face immunostaining revealed that disturbed flow patterns (generated by a constrictive cuff) elevated RelA expression in murine carotid arteries via JNK1. Positron emission tomography and en face staining revealed that disturbed flow enhanced 18F-fluorodeoxyglucose uptake (a marker of inflammation) and accumulation of CD68-positive inflammatory cells in arteries via JNK1. We conclude that disturbed flow promotes arterial inflammation via a novel JNK-NF-κB cross-talk. The duration of RelA nuclear localisation is an important determinant of the magnitude and specificity of target gene expression. En face staining revealed that RelA rapidly accumulated in the nucleus upon LPS stimulation in ECs at both athero-protected and athero-susceptible sites. RelA was exported from the nucleus to the cytoplasm in response to prolonged stimulation in the athero-protected region but not in the athero-susceptible region. The duration of RelA nuclear localisation was suppressed by histone deacetylases which displayed higher activity at the protected site compared to the susceptible site. Overall, our findings reveal that ECs at athero-susceptible sites are primed for inflammatory activation via complementary mechanisms that enhance both the expression and the activity of NF-κB transcription factors.

Atherosclerosis occurs in a spatially heterogeneous fashion within the arterial system. The patchy nature of the disease is thought to reflect spatial variation in haemodynamic factors such as wall shear stress (WSS) and in the concentration of vascular species. This thesis numerically investigates blood flow and vascular transport of the atheroprotective agent nitric oxide (NO) using a spectral/hp element method. Firstly, NO distribution is analysed within a parallel-plate flow chamber coated with endothelial cells. Contrary to the accepted hypothesis that NO concentration increases with WSS (as does NO production), it is observed that NO concentration depends on WSS in a non-monotonic fashion. Furthermore, these results emphasise the effect of convection on in vitro NO transport, which has been overlooked or misinterpreted in most previous computational studies. Secondly, blood flow and NO transport are investigated within a detailed representation of the rabbit thoracic aorta and its branches, which was reconstructed using high resolution computed tomography (CT) scan data of a vascular corrosion cast. The computed WSS distribution exhibits significant spatial heterogeneity, reflecting the complexity of the blood flow. In particular it is observed that two Dean-type vortices (associated with a skewed velocity profile) form in the aortic arch and propagate along the descending aorta. This results in streaks of WSS similar in nature to the fatty streaks of early stage atherosclerosis observed in mature rabbits. This finding provides further support for the hypothesis that blood flow mediates atherogenesis. The vascular distribution of NO is found to depend significantly on the relationship between NO production and WSS. Furthermore, it is concluded that mechanisms preventing NO consumption by haemoglobin (such as diffusional barriers) must exist in order for NO to exert its atheroprotective action. This study represents the first effort to model NO transport in a realistic representation of the major arteries.