Endothelial cells (EC) maintain homeostasis through the tightly controlled balance between expression of protective genes and repression of pro-inflammatory genes, and loss of this balance can cause endothelial dysfunction, leading to inflammatory diseases including atherosclerosis. We have previously shown that the ETS transcription factor Erg is involved in maintaining EC homeostasis through transactivation of genes involved in key functions including angiogenesis, migration and survival. In addition to the role for Erg as a transcriptional activator, recent genome wide gene expression analysis has also highlighted a role for Erg in the repression of multiple pro-inflammatory genes. In this Thesis I describe a novel mechanism for Erg-mediated repression of these pro-inflammatory genes using ICAM-1 as a model. We identify two ETS binding sites (EBS) within the ICAM-1 promoter (EBS-118 and -181) which are required for Erg mediated repression. One of these EBS is within a functional NF-κB binding site. We show that the increase in ICAM-1 expression upon Erg inhibition is NF-κB dependent, and that Erg prevents NF-κB p65 from binding to the ICAM-1 promoter, suggesting a direct mechanism of interference. Gene Set Enrichment Analysis (GSEA) of transcriptome profiles of Erg and NF-κB dependent genes, together with chromatin immunoprecipitation (ChIP) studies, reveals that this mechanism is common to other pro-inflammatory genes, including cIAP2 and IL8. We investigate the role of chromatin modifying enzymes and histone modifications in Erg-mediated repression and show that in quiescent EC the ICAM-1 promoter is also bound by the histone methyltransferase ESET, and by HDAC1, both indicators of a repressed chromatin structure. Moreover, in silico data on histone modifications suggest that in quiescent EC the ICAM-1 promoter is in a repressed conformation. The results from this Thesis suggest that Erg acts as a gatekeeper to inhibit transactivation of pro-inflammatory genes in quiescent EC, providing an important barrier to protect against inappropriate endothelial activation.

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The purpose of the following studies was to explore the effect of systemic vascular and endothelial dysfunction upon the ocular circulation and functionality of the retina. There are 6 principal sections to the present work. Retinal vessel activity in smokers and non-smokers: the principal findings of this work were: chronic smoking affects retinal vessel motion at baseline and during stimulation with flickering light; chronic smoking leads to a vaso-constrictory shift in retinal arteriolar reactivity to flicker; retinal arteriolar elasticity is decreased in chronic smokers. The effect of acute smoking on retinal vessel dynamics in smokers and non-smokers: the principal finding of this work was that retinal reactivity in chronic smokers is blunted when exposed to clicker light provocation immediately after smoking one cigarette. Ocular blood flow in coronary artery disease: The principal findings of this work were: retrobulbar and retinal blood flow is preserved in CAD patients, despite a change pulse wave transmission; arterial retinal response to flickering light provocation is significantly delayed in CAD patients; retinal venular diameters are significantly dilated in CAD patients. Autonomic nervous system function and peripheral circulation in CAD: The principal findings in this work were: CAD patients demonstrate a sympathetic overdrive during a 24 period; a delay in peripheral vascular reactivity (nail-fold capillaries) as observed in patients suffering from CAD could be caused by either arteriosclerotic changes of the vascular walls or due to systemic haemodynamic changes. Visual function in CAD: The principal findings in this work were: overall visual function in CAD patients is preserved, despite a decrease in contrast sensitivity; applying a filtering technique selecting those with greater coefficient of variance which in turn represents a decrease in reliability, some patients appear to have an impaired visual function as assessed using FDT visual field evaluation. Multiple functional, structural and biochemical vascular endothelial dysfunctions in patients suffering from CAD: relationships and possible implications: The principal findings of this work were: BMI significantly correlated with vWF (a marker of endothelial function) in CAD patients. Retinal vascular reactivity showed a significant correlation with peripheral reactivity parameters in controls which lacked in the CAD group and could reflect a loss in vascular endothelial integrity; visual field parameters as assessed by frequency doubling technology were strongly related with systemic vascular elasticity (ambulatory arterial stiffness index) in controls but not CAD patients.

Cannabinoids cause both acute and time-dependent vasodilation/vasorelaxation in a range of vascular beds. The vascular effect of cannabinoids is dependent on the cannabinoid ligand, the species studied and the vascular bed used. To date, there have been four studies that have characterised the effects of cannabinoids in humans. Therefore, the aim of this thesis was to characterise the pharmacological effects of cannabinoids in the human mesenteric artery. Written Informed consent was granted for the use of mesenteric arteries collected from patients at the Royal Derby Hospital. Arteries were dissected from mesenteric tissue and mounted on a Mulvany-Halpern myograph. Arteries were contracted using U46619 and endothelin-1. Concentration-response curves were carried out to the phytocannabinoids THC and CBD; the endocannabinoids AEA and 2-AG; the synthetic cannabinolds CP55,940 and HU-308. The underlying mechanisms of action were assessed using receptor antagonism, enzyme inhibition, endothelium denudation and ion channel manipulation. Experiments to probe the potential for cannabinoids to cause time-dependent vasorelaxation of human mesenteric arteries were also carried out. Post-hoc analysis was conducted on all acute vasorelaxation responses to assess the potential influence of patient characteristics/disease state on cannabinoid responses. All cannabinoids tested, with the exception of HU-308, caused concentration-dependent vasorelaxation of human mesenteric arteries. The synthetic cannabinoid CP55,940 had the greatest Rmax of all the cannabinoids tested. 2-AG had the greatest Rmax of the endocannabinoids tested and CBD had the greatest Rmax of the phytocannabinoids tested. Compared to animal models, cannabinoid efficacy was reduced in human mesenteric arteries. The vasorelaxant effects of 2-AG were mediated through COX-1 metabolism, prostanoid receptor activation (EP4 and IP) and ion channel modulation. The mechanisms underpinning CBD-induced vasorelaxation were CBl and TRPV1 receptor activation, NO release, the endothelium and ion channel modulation. Vasorelaxant responses to AEA were inhibited by antagonism of the CB1 receptor and a putative cannabinoid receptor located on the endothelium (CBe), nitric oxide synthase inhibition and endothelium denudation. Cannabinoid responses were reduced in patients with cardiovascular diseases/disease risk factors including ischaemic heart disease, type-2 diabetes and hypercholesterolemia. Endocannabinoid responses were reduced in patients taking NSAID medication, with some reductions in responses seen to other medication including statins and beta-blockers. CBD and AEA were tested for time-dependent vasorelaxation. Both CBD and AEA were able to cause vasorelaxatlon that gradually increased over time, this was not mediated by the PPARy receptor. This thesis concludes that cannabinoids are able to modulate vascular tone in isolated human mesenteric arteries, and this may be blunted in patients with cardiovascular disease. Furthermore, this thesis presents data suggesting that differences exist between human and animal arterial responses to cannabinoids.

Perineal descent (PD) is a sign of connective tissue weakness of the pelvic floor, it can be measured mechanically or radiologically. Joint hypermobility can be a sign of a generalised connective tissue abnormality, there is an increased incidence of pelvic organ prolapse and faecal incontinence amongst patients with heritable connective tissues diseases. To explore the relevance of PD and the relationship between connective tissue abnormality and pelvic floor dysfunction five studies were performed.A new mechanical device for the measurement of PD, the laser commode, and the established mechanical device, the perineometer were compared to the current gold standard method of measurement, defaecating proctography in 68 subjects. The laser commode provided a mean overall PD measurement closer to that of proctography than the perineometer but the repeatability and reproducibility of the measurements were not accurate enough for the laser commode to be used either in the subsequent parts of this research project or in a clinical setting.Perineal descent was measured using proctography and joint hypermobility was measured using the Beighton score in 70 females with pelvic floor dysfunction. No correlation was found between PD and joint mobility.A review of 323 proctograms of females with pelvic floor dysfunction found an association between PD and rectal prolapse but no association between either PD and rectocele formation or PD and rectal intussusception. The Pelvic Floor Distress Inventory questionnaires of 133 females were correlated with their proctography findings. There was no association between PD and any of the clinical symptoms. Biopsies from the rectus sheath and pelvic floor fascia of 19 females with rectal prolapse were compared to those of 8 normal controls. There was no difference in collagen or elastin content between the groups but participant numbers were small. The pelvic floor fascia of the rectal prolapse group showed a higher percentage of well organised elastin than that of the control group but this did not reach statistical significance. Perineal descent does not appear to be a consistent indicator of severe pelvic floor connective tissue abnormality or injury. This study has furthered our understanding of perineal descent and the relationships between this finding and other pelvic floor disorders caused by connective tissue weakness. Future work will focus on further histological analysis of tissue from patients with rectal prolapse in combination with the use of more sensitive methods to establish the presence of an underlying connective tissue abnormality.

Les objectifs de ce travail expérimental étaient : 1. Évaluer la place de la lactatémie capillaire comme dispositif de monitorage de l’ischémie et de la reperfusion tissulaire. 2. Comparer le suivi local du taux de lactate, au niveau du compartiment ischémié, par rapport au taux systémique du lactate lors des l’ischémie de membre, l’ischémie intestinale ainsi que durant le choc hémorragique. La mesure capillaire du taux de lactate pourrait permettre: 1. d’affirmer l’existence d’une ischémie tissulaire régionale. Le taux systémique du lactate ne permet ce diagnostic, 2. de confirmer l’efficacité d’une reperfusion au niveau d’un membre préalablement ischémique tandis que la mesure systémique des lactates ne le permet pas. L’affirmation de l’efficacité de la reperfusion et de sa constance, par une mesure aussi simple que la lactatémie capillaire compartimentale représente une avancée majeure en comparaison avec les autres méthodes existantes. La mesure capillaire du taux de lactate systémique dans une situation d’hypoperfusion par choc hémorragique n’est pas associée à augmentation du taux de lactate intra-musculaire. / Our objectives for this experimental work were: 1. Assessment of capillary lactate for tissue ischemia and reperfusion monitoring. 2. Compare the local and systemic capillary lactate time course during compartmental ischemia insults like limb ischemia, intestinal ischemia or during hemorrhagic shock. The capillary measurement of the lactate in IR could be interesting for: 1. Assessing a limb or intestinal tissue ischemia. On the opposite, the systemic measurement could not assess this diagnosis. 2. Assessing the efficiency of a limb reperfusion. On the opposite, the systemic measurement could not assess this diagnosis. The systemic capillary lactate measurement during haemorrhagic shock related hypo perfusion could not be associated with an increase in intra muscular lactate.

La déstabilisation de la plaque d’athérosclérose reste de nos jours un problème majeur, malgré les progrès récents dans sa compréhension. Les neutrophiles sont des acteurs puissants de l’immunité innée capables d’altérer les plaques. Un chimio-attractant majeur des neutrophiles, le leucotriène B4, pourrait être un des contributeurs potentiels de la déstabilisation des plaques en particulier dans l’endotoxémie, elle-même associée aux accidents cardiovasculaires. L’objectif de ce travail a été de définir le rôle du leucotriène B4 dans l’attraction des neutrophiles dans la plaque au cours de l’endotoxémie et de déterminer si les neutrophiles peuvent basculer l’équilibre qui maintient les plaques stables. Nous avons montré que le recrutement des neutrophiles médié par le leucotriène B4 a un impact délétère sur la stabilité des plaques au cours de l’endotoxémie en favorisant l’apoptose et la dégradation de fibres matricielles. En conclusion, cette étude ouvre la voie vers de nouvelles approches thérapeutiques visant à cibler l’axe leucotriène-neutrophiles dans la maladie athérosclérotique. / Atherosclerotic plaque destabilisation remains an important issue, in spite of the recent advances in its comprehension. Neutrophils are powerful innate immune actors capable of altering plaques. In this context, the leukotriene B4, one of the main chemoattractants of neutrophils, has been proposed as a potential contributor to plaque destabilisation. A particular context in which these two actors are closely linked is endotoxemia, itself associated with plaque destabilisation This work was aimed at determining whether leukotriene B4 plays a role in the chemoattraction of neutrophils in plaques during endotoxemia and at assessing whether neutrophils can tip the balance which maintains plaques stable. We have herein evidenced that the recruitment of neutrophils mediated by leukotriene B4 has a deleterious impact upon plaque stability during endotoxemia by promoting apoptosis and degrading matrix fibres. In conclusion, this study paves the way to novel therapeutic approaches aimed at targeting the axis leukotriene-neutrophil in atherosclerotic disease.

L’ischémie d’exercice caractérisée par l’incapacité du système circulatoire de faire face à l’augmentation des besoins en substrats énergétiques et en oxygène nécessaire à dégrader ces substrats. La claudication à la marche est une cause majeure de limitation fonctionnelle.Il existe de nombres techniques d'évaluation de la macrocirculation et de la microcirculation permettant d'évaluer la réponse endothéliale dépendante(iontophorèse, test au garrot, modification de température locale) avec un enregistrement laser Doppler ou Speckle.Si les techniques ultrasonores permettent d’explorer la présence de lésions, elles rendent mal compte de la collatéralité. Ces techniques peu applicables à l’exercice et nécessitent d’être améliorées pour être applicables à l’effort. L’ischémie artérielle, entraîne une souffrance cellulaire avec métabolisme anaérobie, réversible à l’arrêt de l’exercice. En cas de développement de circulation collatérale suffisante, l’ischémie est alors incomplète, la souffrance des tissus est modérée et réversible rapidement. La recherche de pathologie artérielle au reposa été largement étudiée, nous nous intéressons dans nos études à la pathologie artérielle à l’effort, mais aussi aux phénomènes physiopathologiques susceptibles d’interférer avec la fonctionnalité musculaire (hypoxémie induite par l’exercice). La mesure de la pression partielle transcutanée en oxygène (TcpO2) à l’exercice permet d’estimer en cours de l’exercice l’importance de l’ischémie, segment de membre par segment de membre,de façon bilatérale et continue. Par cette nouvelle technique nous tentons d’explorer la physiopathologie de l’ischémie vasculaire à l’exercice. / Exercise ischemia is characterized by the inability of the circulatory system to fulfil the increased need forenergy substrates and the oxygen needed for substrates’ metabolism. Claudication is a major cause of functional limitation. There are several methods for assessing macrocirculation (mainly ultrasound imaging) and microcirculation (iontophoresis, tourniquet test, local temperature modification with Laser or Speckle recording. If ultrasound techniques can explore the occurrence of lesions is not optimal to evaluate the hemodynamic consequences because pressure measurements do not necessarily correlate with flow impairment. Laser techniques are not appropriate during exercise tests and need to be improved to be applicable. During exercise the severity of arterial ischemia depends on collateral circulation. Further ischemia is reversible at the end of exercise. Although research of restingarterial ischemia has been extensively studied few isknown in arterial ischemia during exercise, but al soin other physiopathological dysfunctions that may interfere with muscle function (exercise-inducedhypoxemia). The measurement of the transcutaneous oxygen partial pressure (TcpO2) during exercise estimates during exercise the importance of ischemia, limb segment by limbsegment, bilaterally and continuously. With this new technique we try to investigate the Physiopathology of vascular ischemia during exercise.