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The vascular endothelium contributes to the maintenance of vascular health by regulating vascular tone and leukocyte adhesion, amongst others. The vasoregulatory actions of the endothelium are mediated through coordinated release of vasodilators such as nitric oxide (NO) and prostacyclin, and vasoconstrictors such as endothelin-1 and thromboxane A2. Endothelial NO is the principal vasodilator in the vasculature and is produced by endothelial nitric oxide synthase (eNOS). Insulin is a vasoactive hormone that exerts its vasodilatory effects through eNOS-mediated NO production. Endothelial function is impaired in a number of disorders, including insulin resistance, diabetes and atherosclerosis, leading to dysregulated vasodilation as well as increased monocyte adhesion and plaque formation (atherosclerosis). The underlying molecular mechanisms leading to endothelial dysfunction are still in question. The work presented in this thesis addressed this question by investigating how insulin signalling and eNOS-mediated NO and superoxide production in human vascular endothelial cells are affected under experimental hyperinsulinaemia (chapter 3) and experimental hyperglycaemia (chapter 4). Atherogenic processes in human aortic endothelial cells (HAEC) were investigated by assessing monocyte adhesion under experimental hyperinsulinaemia (chapter 3), and by determining the contribution of NO and AMP-dependent kinase (AMPK) activity to the regulation of endothelial chemokine production (chapter 6). The potential of insulin to modify the subcellular distribution of eNOS was investigated in chapter 5. Clinical hyperinsulinaemia correlates with attenuated NO-mediated vasodilation, but it is not clear how hyperinsulinaemia impairs eNOS-mediated NO production. The present study modelled hyperinsulinaemia in HAEC and demonstrated a blunted response of hyperinsulinaemic cells to Ca2+-stimulated, but not insulin-stimulated eNOS-mediated NO synthesis. To address the underlying mechanisms responsible, the protein expression levels of components of the metabolic and mitogenic insulin signalling pathways, and of the metabolic energy sensor, AMPK, were quantified. Experimental hyperinsulinaemia slightly and non-significantly increased basal and insulin-stimulated eNOSS1177 phosphorylation in a time-dependent manner, and the levels of eNOST495 increased following acute insulin stimulation under these conditions. No marked dysregulation of individual insulin signalling pathway components was identified as a potential cause, but increased activating AMPKT172 phosphorylation was found to be a potential cause of increased unstimulated eNOSS1177 phosphorylation under experimental hyperinsulinaemia. Monocyte adhesion to hyperinsulinaemic HAEC did not differ from control HAEC, indicating that experimental hyperinsulinaemia did not act as a proatherogenic factor in the present study. Overt diabetes was simulated by experimental hyperglycaemia in human umbilical vein endothelial cells (HUVEC) and its effect on insulin-stimulated eNOS phosphorylation and endothelial superoxide production assessed. Insulin tended to stimulate phosphorylation of eNOSS615 and eNOSS1177, and decrease phosphorylation of eNOSS114, eNOST495 and eNOSS633 under control conditions. Experimental hyperglycaemia slightly reduced basal phosphorylation of Ser633 and significantly reduced insulin-stimulated phosphorylation of Ser114, but mildly increased basal Ser615 phosphorylation, indicating some dysregulation of eNOS phosphorylation. The upstream components of the metabolic insulin signalling pathway were not impaired in hyperglycaemic conditions. The subcellular localisation of eNOS is thought to have implications for its function. This study showed that eNOS localises to the plasma membrane, the nucleus, the cytoplasm and, primarily, the perinuclear area of HAEC. Insulin stimulation did not affect this distribution. Phospho-eNOS species were found primarily at the plasma membrane, and insulin may modulate the abundance of an intracellular eNOST495 species. Previous work in our laboratory on AMPK-mediated reduction of adhesion molecule expression has lead to the investigation of AMPK- and NO-mediated regulation of chemokine production in the present study. Inhibition of NO synthesis increased the production of monocyte chemoattractant protein (MCP)-1 in HAEC. AMPK activity downregulated TNFα-stimulated MCP-1 expression, and this was NO-dependent in the short-term, but may be NO-independent during prolonged AMPK activation. These data implicate NO and AMPK as antiatherogenic mediators in vascular endothelial cells in vitro. Taken together, the data in this thesis provide further insight into some of the molecular mechanisms involved in endothelial function and their response to hyperinsulinaemia, hyperglycaemia and proatherogenic stimulation.

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Dans la dysfonction endothéliale, le compartiment endothélial circulant joue simultanément le rôle d’acteur impliqué dans la régénération du tissu lésé et celui d’indicateur de l’état d’altération ou de régénération de l’endothélium. Dans l’artérite oblitérante des membres inférieurs (AOMI), l’un des axes de recherche porte sur le développement d’un produit de thérapie cellulaire capable d’induire la formation de néo-Vaisseaux. Face à la difficulté d’obtenir et d`amplifier des cellules progénitrices endothéliales (CPE) chez l’adulte sain, et a fortiori chez le patient, l’une des hypothèses laisse envisager le recours à d’autres types cellulaires ayant des propriétés vasculogéniques. Chez patients atteints de maladies cardiovasculaires, et d’AOMI en particulier, les cellules mononuclées de moelle osseuse et les CPE montrent des propriétés angiogéniques diminuées. Nous avons mis en évidence la capacité des cellules souches mésenchymateuses (CSM) isolées de patients atteints d’AOMI à induire une reperfusion, par recrutement de cellules endothéliales in situ, avec la même efficacité que celles de donneurs sains. Les CSM ne se différencient pas en cellules endothéliales mais agissent par paracrinie. La seconde hypothèse d’obtention d’un produit de thérapie cellulaire autologue angiogène est de trier des cellules plus immatures que les CPE afin de les différencier secondairement vers la lignée endothéliale à l’image du modèle pathologique de la cellule souche d’hémangiome CD133+ qui laisse envisager les Very Small Embryonic Like stem cells (VSEL), cellules souches multipotentes CD133+, comme un candidat de cellules post-Natales à potentiel vasculaire. Nous avons dérivés, en culture en conditions angiogéniques, des VSEL qui acquièrent un phénotype mésenchymateux mais présentent un profil sécrétoire proche de celui des CPE. Les VSEL favorisent la revascularisation post-Ischémique et acquièrent un phénotype endothélial in vitro et in vivo suggérant que les VSEL peuvent être à l’origine de la lignée endothéliale. Les VSEL se présentent également comme un biomarqueur de la dysfonction endothéliale mobilisé de la moelle osseuse (MO) vers le sang périphérique (PB) chez les patients souffrant d’AOMI. Les biomarqueurs cellulaires circulants représentent non seulement des marqueurs non invasifs de l’endothélium mais peuvent également apporter des informations utiles pour le diagnostic, le pronostic et le suivi thérapeutique des patients souffrant de pathologies associées à une dysfonction endothéliale. Une modification du nombre de CPE et de cellules endothéliales circulantes (CEC) dans la circulation a été rapportée dans différentes situations pathologiques respectivement associées à une régénération et une altération endothéliale telle l’augmentation du taux de CEC chez des patients présentant une hypertension artérielle pulmonaire (HTAP). La technique de référence pour le dénombrement des CEC dans le sang périphérique est l’immunoséparation magnétique (IMS). Cette méthode non automatisée et chronophage, repose sur l’énumération par microscopie à fluorescence des cellules CD146+ préalablement isolées. Bien que reproductible, cette numération est soumise à de nombreux biais de quantification, difficile à mettre en oeuvre et sujette à interprétation. La mise au point d’une méthode de détection automatisée des CEC par cytométrie à focalisation acoustique (AFC) s’est montrée fiable et robuste, dans une cohorte de patients atteints d’HTAP traitée ou non, constituant une alternative pertinente à l’analyse par microscopie. L’ensemble de ces travaux ouvre donc de nouvelles perspectives dans la détection des biomarqueurs cellulaires circulants impliqués dans la dysfonction endothéliale, proposant les VSEL comme nouvel acteur vasculogénique. / In endothelial dysfunction, circulating endothelial compartment simultaneously plays the role of actor involved in the regeneration of injured tissue and reflects endothelium state. In peripheral arterial disease (PAD), one of the research areas is the development of a cellular therapy product capable of inducing the formation of neo-Vessels. Faced with the difficulty to obtain and amplify endothelial progenitor cells (EPC) in adults, one of the assumptions lets consider the use of other cell types with vasculogenic properties. In patients with cardiovascular disease, and PAD in particular, bone marrow mononuclear cells and EPC show reduced angiogenic properties. We have demonstrated the ability of isolated mesenchymal stem cells (MSCs) from PAD patients to induce reperfusion by recruitment of endothelial cells in situ, with the same efficiency as that of healthy donors MSCs. MSCs do not differentiate into endothelial cells but act by paracrine. The second hypothesis of obtaining an autologous angiogenic cell therapy product is to sort cells more immature than the CPE and to differentiate them secondarily into endothelial lineage as the pathological cell model of hemangioma stem cells CD133 + which lets consider the Very Small Embryonic like stem cells (VSEL), CD133 + multipotent stem cells as a potential candidate of postnatal vascular cell. We have derived and cultured in angiogenic conditions VSEL that acquired a mesenchymal phenotype but exhibited a secretory profile similar to that of EPC. VSEL promote post-Ischemic revascularization and acquire an endothelial phenotype in vitro and in vivo suggesting that VSEL may be responsible for the endothelial lineage. VSEL also appear as a biomarker of endothelial dysfunction mobilized from bone marrow (BM) to peripheral blood (PB) in patients with PAD. Cellular circulating biomarkers are not only non-Invasive markers of endothelium but can also provide useful information for the diagnosis, prognosis and therapeutic monitoring of patients with endothelial dysfunction associated pathologies. Changing the number of EPC and circulating endothelial cells (CEC) in the circulation has been reported in different pathological situations respectively associated with endothelial regeneration and alteration such as the increase of CEC in patients with pulmonary arterial hypertension (PAH). The reference technique for the enumeration of CEC in peripheral blood is magnetic immunoseparation (IMS). This non-Automated and time-Consuming method, based on the enumeration by fluorescence microscopy of CD146 + cells isolated. Although reproducible, this count is subject to many through quantification, difficult to implement and subject to interpretation. The development of an acoustic focusing cytometry (AFC) method for automated detection of CEC has proved reliable and robust results, in a cohort of patients with PAH treated or not, constituting a relevant alternative analysis to microscopy. All of this work opens new perspectives in the detection of cellular circulating biomarkers involved in endothelial dysfunction, suggesting VSEL as new vasculogenic actor.