AbstractAtherosclerosis is an arterial inflammatory and degenerative disease characterized by a subendothelial accumulation of lipids, leading to the formation of an atheroma. The rupture of the atheroma is associated with clinical outcomes such as myocardial infarction and thrombosis. Atherosclerotic lesions develop preferentially in arterial regions subjected to disturbed or low shear stress, which represents the tangential frictional force of the blood flow at the surface of endothelial cells. These local hemodynamic conditions promote the intimal accumulation of low-density lipoproteins (LDL), especially in case of hypercholesterolemia. In the intima, LDL are modified, notably through their oxidation, and initiate an inflammatory response, promoting the recruitment of circulating monocytes by endothelial cells. The differentiation of monocytes into macrophages and the excessive and uncontrolled internalization of modified LDL lead to the formation of lipid-laden macrophages, also termed foam cells. The apoptotic death of foam cells contributes to the formation of the necrotic core and to the development of the atherosclerotic plaque. The physiological oxidizing agents implicated in LDL oxidation in vivo are still unambiguously identified. In vitro, LDL are commonly oxidized using copper sulfate, leading to the formation of LDL modified both at the lipid and protein moieties. However, the physiological relevance of copper in LDL oxidation is more and more called in question. In this way, alternative and more physiological methods of LDL oxidation have emerged, such as myeloperoxidase. In this study, we evaluated the effects of native, copper-oxidized LDL (OxLDL) and myeloperoxidase-oxidized LDL (MoxLDL) on the activation of the anti-oxidant and anti-inflammatory Nrf2-dependent signalling pathway in macrophages and endothelial cells: two cell types largely involved in the development of atherosclerotic lesions. Moreover, the MoxLDL used in this study are exclusively modified on the protein moiety. Our results showed that both types of oxidized LDL activate the Nrf2 transcription factor in macrophages, contrary to native LDL. Moreover, the activation of the Nrf2 pathway triggered by the incubation with MoxLDL is more important than the activation triggered by the incubation with OxLDL. This differential activation can be explained, at least partly, by a more important production of reactive oxygen species (ROS) in cells exposed to MoxLDL through a specific pathway implicating cytosolic PLA2. Moreover, we showed that the non-oxidized lipids of MoxLDL are certainly involved in this ROS production. On the other hand, MoxLDL are not able to induce the activation of the Nrf2 pathway in endothelial cells, in spite of the induction of an important intracellular oxidative stress. On the contrary, the incubation of endothelial cells with OxLDL induces the activation of the Nrf2 pathway, but through a ROS-independent pathway, involving PKCs. In vivo, endothelial cells are constantly exposed to local hemodynamic forces induced by blood flow. In this study, we evaluated the effects of a high or low laminar shear stress, representative of the hemodynamic conditions in arterial regions respectively protected and susceptible to the development of atherosclerotic lesion, on the expression of genes in cultured endothelial cells. Using micro-fluidic cards in Real-time PCR, we showed that a low laminar shear stress globally induces the expression of pro-atherogenic genes in endothelial cells, whereas cells exposed to a high laminar shear stress exhibit an anti-atherogenic expression profile. Moreover, a high laminar shear stress induces the nuclear translocation of the Nrf2 transcription factor. Interestingly, the addition of oxidized LDL disturbs the endothelial response to a high shear stress. These preliminary results suggest that the study of the effects of risk factors on endothelial cells should be performed in relevant hemodynamic conditions, in order to better understand their pro-atherogenic role in vivo. In conclusion, the results of this thesis notably underline that macrophages and endothelial cells are able to discriminate copper-oxidized LDL, modified both at the lipid and protein moieties, of myeloperoxidase-oxidized LDL, more physiologically relevant and only modified on the protein fraction, and respond very differently to these two types of oxidized LDL. This reality should be kept in mind in order to better understand the physiological mechanisms implicated in atherogenesis, but also to suggest intervention studies in mice, and maybe new therapies in the future, complementary to the battery of therapeutics currently available.
|Date of Award||13 May 2011|
|Supervisor||Martine Raes (Supervisor), Jean-Claude MAZIERES (Jury), Jean Ducobu (Jury), Yves Poumay (Jury), Thierry Arnould (Jury) & Xavier De Bolle (President)|
- Lipoprotéines de faible densité
- sulfate de cuivre
Étude comparative des effets des LDL oxydées avec du sulfate de cuivre ou de la myéloperoxydase sur des macrophages et des cellules endothéliales en culture – Importance de la voie de signalisation Nrf2
Calay, D. (Author). 13 May 2011
Student thesis: Doc types › Doctor of Sciences