Atherosclerosis is a complex inflammation condition involving the accumulation of lipid-filled macrophages within the artery wall. Progression of the initial fatty streak to an advanced atherosclerotic plaque is characterized by the development of a necrotic core region containing cholesterol and dead cells. The oxidation of low-density lipoprotein (LDL) to oxidized LDL (oxLDL) and its subsequent uptake by macrophages to form foam cells are the key process in plaque formation. OxLDL is found within atherosclerotic plaque, and it is cytotoxic to a range of cells including macrophages through the generation of reactive oxygen species (ROS) and induction of oxidative stress. The aim of this study was to examine the cytotoxic effects of oxLDL to U937 human monocyte-like cells. OxLDL caused a rapid concentration-dependent cell viability loss in U937 cells within 6 hours. The progression of oxLDL-induced cell death was found to be strongly correlated with the intracellular ROS production and intracellular glutathione (GSH) loss. OxLDL also caused a rapid loss of intracellular aconitase activity, indicating the impairment of the cellular metabolic function. The cytosolic calcium ion (Ca²⁺) level was also elevated by oxLDL, which could be from both intra- and extra-cellular sources. OxLDL also activated plasma membrane superoxide generation complex NADPH oxidase (NOX), and the progression of oxLDL-induced NOX activation was correlated with oxLDL-mediated ROS production, suggesting NOX is the major source of ROS. Further investigations using NOX inhibitors apocynin or diphenyleneiodonium (DPI) found that inhibition of NOX prevented oxLDL-induced cell viability loss, ROS production, GSH loss and aconitase activity decrease. The cytosolic Ca²⁺ elevation caused by oxLDL was also suppressed slightly by inhibiting NOX activity. These results clearly show that NOX is the major site of oxidative stress upon oxLDL activation, contributing to the oxLDL-induced cell death. This study also examined the protective effect of 7,8-dihydroneopterin (7,8-NP) on oxLDL-induced oxidative stress. 7,8-NP dramatically protected cells from oxLDL-induced cell viability loss, ROS generation and aconitase activity loss. 7,8-NP also inhibited oxLDL-induced cytosolic Ca²⁺ influx particularly after 3 hours. 7,8-NP did not inhibited mitochondrial aconitase activity decrease caused by oxLDL, nor inhibited mitochondrial ROS production. This indicates the protective effect of 7,8-NP against oxLDL damage could primarily in cytoplasm. The failure of 7,8-NP protection from oxLDL activating NOX suggests that the protection of 7,8-NP against oxLDL-induced oxidative stress was not due to the inhibition of NOX activation, but by radical scavenging activity of the NOX products.

Aortic dissection is a detrimental disease with a high mortality. However, the mechanisms regulating the susceptibility to aortic dissection remain unknown. We hypothesize that endothelial oxidative stress due to the activation of the reactive oxygen species (ROS)-generating Nox2 enzyme may play an important role in the development of aortic dissection. To investigate this, we generated transgenic mice (C57BL/6J background) with endothelial specific over-expression of Nox2 (Nox2 Tg) under the control of a tie-2 promoter. Expression of the human Nox2 transgene was confirmed by qRT-PCR to be found only in endothelial cells (EC) isolated from transgenic mice, and not in Wt EC or vascular smooth muscle cells (VSMC) and macrophages isolated from either genotype. Wild-type (Wt) littermates and Nox2 Tg male mice (22-24 weeks old, n=11) were treated with saline or Ang II (1mg/kg/day) via subcutaneous mini-pump for 28 days. There was no significant difference in the pressor responses to Ang II between Wt and Nox2 Tg mice (Wt 121±7mmHg vs. Nox2-Tg 122±6mmHg). However, 5/11 Nox2 Tg mice developed aortic dissections compared to 0/11 Wt mice (P<0.05). Immunohistochemistry revealed significant increases in endothelial VCAM-1 expression, MMP activity and CD45+ inflammatory cell recruitment in the aortas of Nox2 Tg mice after 5 days of Ang II infusion. Inflammatory cell recruitment was confirmed by FACS analysis of cells from digested aortas (P<0.05). Explanted aortas from Nox2-Tg mice had significantly greater secreted pro-inflammatory cytokine, Cyclophilin A (CypA) both at baseline and after 5 days of Ang II infusion compared to Wt littermates. Compared to primary Wt EC and VSMC, Nox2-Tg primary EC, but not primary VSMC, had increased ROS production which was accompanied by increased endothelial CypA secretion and ERK1/2 activation. Furthermore, conditioned media from Nox2-Tg EC induced greater ERK1/2 phosphorylation compared to conditioned media from Wt controls. Knockdown of CypA from sEND.1 endothelial conditioned media by siRNA knockdown abolished VSMC Erk1/2 phosphorylation. In conclusion, we demonstrate for the first time that a specific increase in endothelial ROS through the over-expression of Nox2 was sufficient to induce aortic dissection in response to Ang II stimulation. Endothelial secreted CypA could be the signalling mechanism by which increased endothelial ROS regulates the inflammatory response and the susceptibility to aortic dissection.

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La NADPH oxydase du phagocyte est une enzyme impliquée dans la défense immunitaire contre les pathogènes. Après activation du phagocyte, cette enzyme produit des ions superoxyde par réduction du dioxygène par le NADPH. Elle est constituée de quatre sous- unités cytosolubles (p47phox ; p67phox ; p40phox et Rac), et deux membranaires (gp91 ; p22phox). Son activation fait intervenir un processus complexe qui met en jeu des changements d’interaction entre les protéines la constituant et qui permet l’assemblage des six sous- unités. Afin d’obtenir des informations sur les processus d’assemblage et d’activation, j’ai reconstitué le complexe dans un système cell free à l’aide de protéines recombinantes pour pouvoir contrôler tous les paramètres. Dans ce travail nous avons comparé les modes d’activation de p47phox par phosphorylation, par mutation substitutionelle sérine - aspartate en position S303,S304 et S328 pour mimer la phosphorylation et enfin par addition d’acide arachidonique (AA) activateur connu de l’enzyme in vitro mais aussi in vivo. Bien qu’il ai été montré que ces trois méthodes ouvrent la protéine vers une conformation ayant des propriétés similaires, nous avons trouvé que les effets de ces méthodes d’activation sont significativement différents. Ainsi, les changement de conformation observés par dichroisme circulaire, sont dissemblables. Pour p47phox, l’addition de AA déstructure la protéine. La phosphorylation induit un déplacement bathochrome des bandes de CD qualitativement similaire, alors que les mutations S-D de p47phox provoquent un déplacement opposé. Pour le complexe p47phox-p67phox l’addition d’AA destructure le mélange tandis que la mutation induit relativement peu de changement. Nous avons mesuré les constantes de dissociation Kd du complexe p47phox-p67phox. Alors que pour les protéines « sauvages », le Kd est faible (4±2 nM), les mutations de p47phox ainsi que l’addition d’AA augmentent cette valeur jusqu’à environ 50 nM, montrant une diminution de l’affinité entre p47phox-p67phox. De même, sur le complexe entier, l’effet de la phosphorylation de p47phox est différent de la mutation. Nous avons mesuré les valeurs de EC50 relatives à p67phox pour les différentes formes de p47phox. L’activation de p47phox par phosphorylation diminue l’EC₅₀, alors que les doubles ou triple mutations augmentent sa valeur. Nous avons confirmé que la phosphorylation et la mutation sont insuffisantes pour activer l’enzyme. La présence de AA est indispensable pour le fonctionnement du complexe. L’ordre de fixation des sous unités cytosoliques semble indifférent mais il faut que tous les composants soient présents lors de l’ajout de AA. Enfin, la délétion de p47phox dans la partie C-terminale (aa 343 à 390, domaine d’interaction avec p67phox) il n’y a plus de formation du dimère mais l’enzyme fonctionne normalement. Ces résultats apportent des éléments nouveaux sur le rôle de la dimérisation p47 phox-p67 phox, non indispensable à l’activité du système et sur le rôle mineur de la phosphorylation dans l’activation de la NADPH oxydase in vitro. / The NADPH oxidase of phagocytes is an enzyme involved in the innate defense of organisms against pathogens. After phagocyte activation, this enzyme produces superoxide ions by reduction of dioxygen by NADPH. It is constituted of four cytosolic sub-units (p47phox ; p67phox ; p40phox et Rac) and two membrane proteins (gp91 ; p22phox). Its activation takes place through a complex process that involves protein-protein interaction changes leading to assembly and functionning of the catalytic core. In order to obtain information on this process, I have reconstituted the enzyme in a cell free systeme using recombinant proteins, to be able to fully control all the measurement conditions. In this work, we have compared different activation modes of p47phox i) phosphorylation; ii) substitution serine - aspartate by mutations at positions S303, S304 and S328 to mimic phosphorylation; iii) addition of arachidonic acid (AA), a well known activator molecule in vitro. It has been shown that these three activating methods transform p47phox to an open configuration with similar characteristics. However, we have found that the effects of these methods are significantly different. Indeed, the conformational changes observed by circular dichroism are different. For p47phox, the addition of AA destructures the protein. Its phosphorylation induces a bathochromic displacement of the bands, whereas the mutations S-D lead to an opposite displacement. For the dimer p47phox-p67phox , the addition of AA destructures the proteins while mutations induce hardly no changes. We have measured the dissociation constant Kd of the complex p47phox-p67phox. For wild type proteins, Kd value is low (4±2 nM), while mutations of p47phox as well as addition of AA increase its value up to 50 nM, showing a decrease of affinity between p47phox and p67phox. Moreover, on the whole complex, the effect of phosphorylation of p47phox is different from mutations. We have shown that the EC50 values relative to p67phox are sensitive to the various modifications of p47phox. Phosphorylation of p47phox decreases EC₅₀, while double or triple mutations increase its value. We have confirmed that phosphorylation and mutation are not sufficient to activate the enzyme. The presence of AA is a prerequisite for the functionning of the complex, i.e. production of superoxide. The binding order of the cytosolic proteins seems random but it is necessary that all the components be present during the activation by AA. Finally, deletion of the C terminal part of p47phox (aa 343 to 390, interaction domain with p67phox) leads to the absence of dimer formation but does not affect the enzyme activity. These results bring new information on the role of dimerisation of p47-p67 and on that of phosphorylation in the activation of NADPH oxidase in vitro.