The central role of vascular endothelial growth factor (VEGF) in physiological and pathological angiogenesis makes it attractive both as a therapeutic target for anti-angiogenic drugs in cancer treatment and as a pro-angiogenic cytokine to treat ischemic disease. Currently, it is well established that the VEGF but also other growth factors exert their angiogenic effects partly through the activation of endothelial nitric oxide synthase (eNOS). A better understanding of the VEGF/NO signaling pathway could therefore lead to the identification of new therapeutic targets to impact on angiogenesis.This thesis is based on four different articles, the principal findings of which are summarized here below. First, we demonstrated that, chronologically, endothelial cell exposure to VEGF first led to eNOS dissociation from caveolin (a hallmark of the Ca2+/CaM-mediated activation of eNOS), and then to the interaction of eNOS with the heat shock protein hsp90. We also reported that eNOS-bound hsp90 could recruit VEGF-activated (phosphorylated) Akt to the complex, which in turn could phosphorylate eNOS on the serine 1177. Finally, we found that although the VEGF-induced phosphorylation of eNOS led to a sustained production of NO independently of a maintained increase in intracellular [Ca2+], this late stage of eNOS activation was strictly conditional on the initial VEGF-induced Ca2+-dependent stimulation of the enzyme. These data established the critical temporal sequence of events leading to the sustained activation of eNOS by VEGF and suggested new ways of regulating the production of NO in response to this cytokine through the structural protein caveolin and the ubiquitous chaperone protein, hsp90. A second study identified caveolin and Hsp90 as key players in the proangiogenic action of statins and therefore as potential pharmacological targets to modulate NO-dependent angiogenesis. We found that atorvastatin stabilized endothelial tube formation from both outgrowing and isolated macrovascular ECs cultured in Matrigel through a decrease in caveolin abundance and in its inhibitory interaction with eNOS. In a similar angiogenic assay, microvascular endothelial cells appeared also responsive to statins, not through a decrease in the caveolin pool (which is (too) large in these cells) but via the increased recruitment of hsp90 in the eNOS complex and the associated eNOS phosphorylation on the serine 1177. These data provided new mechanistic insights into the NO-mediated effects of statins and underscored the potential of these drugs and other modulators of hsp90 and caveolin abundance to promote neovascularization in disease states associated or not with atherosclerosis. In a third study, we have reported a net decrease in the ability of cultured ECs expressing recombinant caveolin to migrate and to form capillary like networks (e.g. the crucial steps occurring during the angiogenic process) in presence of VEGF. We then exploited the propensity of cationic lipids to target EC lining tumor blood vessels to transfect tumor-bearing mice in vivo. A dramatic tumor growth delay associated to a decrease in tumor microvessel density in the central core of the tumor was observed in mice transfected with caveolin versus sham-transfected animals. Interestingly, we also found that in the early time after lipofection (e.g. when macroscopic effects on tumor growth were not yet detectable), caveolin expression also impaired NO-dependent tumor blood flow. These findings indicated that besides (before) acting as an anti-angiogenic agent, recombinant caveolin can modulate the endothelium phenotype and impact on the tumor blood flow, both effects leading to a decrease in tumor growth. Finally, we showed that the activation of the VEGF/NO signaling pathway led to the down-regulation of adhesion molecules and to the anergy of endothelial cells. We found, indeed, that the adhesion of human CD8+ lymphocytes on microvascular endothelial cells exposed to TNF-a was dramatically reduced in the presence of VEGF. Interestingly, we also documented that the co-administration of the NOS inhibitor L-NAME or the Hsp90 inhibitor geldanamycin could restore this adhesion to the level originally obtained with TNF-a alone. Finally, we confirmed the key role of NO in the VEGF-mediated effects on the CD8+ adhesion by tipping the balance towards more or less angiogenesis through the transfection of caveolin siRNA or caveolin plasmid, respectively. In these experiments, lymphocyte adhesion appeared directly correlated to the extent of caveolin expression, confirming that the so-called anti-angiogenic strategy can directly impact on the phenotype of (tumor) endothelial cells and instead of (before) killing them, be exploited to potentiate cancer immunotherapy. In conclusion, by dissecting the post-translational regulation of eNOS, we have identified major therapeutic targets, namely caveolin and hsp90, that may be exploited either to block or promote angiogenesis. More particulary, cDNA encoding for these proteins or their mutant form, when combined with adequate mode of delivery, appeared to exert profound effects on the vascular compartment of tumors but also of ischemic tissues.

Lintérêt actuel suscité par la contribution des prostanoïdes en général et par le TXA2 en particulier à lhoméostasie cardio-vasculaire, nous a conduit à mener une série de procédures expérimentales chez le porc. Nous avons ainsi étudié son effet sur la vasomotricité systémique qui se résume en une élévation des résistances vasculaires systémiques (R2) et une diminution de la compliance vasculaire (C). Nous avons également démontré son action pro coagulante et pro inflammatoire dans un processus dischémie myocardique induit par application topique de FeCL3. Dans ce processus, le TXA2 nexerce aucun effet sur lévolution des paramètres hémodynamiques systémiques. Enfin, il joue un rôle mineur dans lischémie-reperfusion myocardique mais il est important de rappeler que dans notre procédure expérimentale, linduction de lischémie se faisait de façon abrupte par application dun clamp coronaire ce qui rend les mécanismes thrombotiques et dont la libération de TXA2 moins prépondérants.Lutilisation d'un modulateur du thromboxane A2, le BM-573, savère efficace dans la prévention des effets de lagoniste du TXA2, lU-46619, sur les résistances vasculaires périphériques. Le BM-573 exerce de plus un effet cardioprotecteur en cas dischémie myocardique, mais aucun effet bénéfique nest observé en cas dischémie-reperfusion myocardique.Le TXA2 joue donc un rôle important dans lhoméostasie cardiovasculaire et intervient avec dautres médiateurs dans la physiopathologie de lhypertension artérielle et de lischémie myocardique. Nous navons cependant pas démontré sa contribution exacte au processus dischémie-reperfusion myocardique / Prostanoids in general and thromboxane A2 in particular appear to play a crucial role in cardiovascular homeostasis. To address this issue, we have carried out a number of experimental procedures in the pigs. We were able to show that the effects of TXA2 on sytemic vascular hemodynamic, consist in an increase of vascular resistance and a decrease of vascular compliance. Additionnaly, TXA2 had a procoagulant and proinflammatory effects in a model of myocardial ischaemia induced by topical application of FECL3 on the left anterior descending coronary artery. No effects were noticed concerning the evolution of systemic hemdynamics parameters. Finally, TXA2 appeared to play a minor role in a myocardial ischaemia-reperfusion model. The use of a TXA2 modulator, BM-573, prevented the systemic vascular effects of TXA2. It also had a cardioprotective effect in the model of myocardial ischaemia, but failed to prevent reperfusion injury in acutely ischemic pigs. We conclude that TXA2 plays an important role in cardiovascular homeostasis and that it intervenes along with others mediators in the physiopathology of hypertension and myocardial ischaemia.