Clinical outcomes of atherosclerosis are often due to plaque rupture. This phenomenon tends to occur in developed and unstable plaques and is favoured by death of macrophage-derived foam cells. During plaque development the death of foam cells also contributes to the formation of the necrotic core and a large necrotic core is one of the features of unstable plaques. As the death mechanisms of foams cells contribute to plaque development as well as to clinical outcomes, it is a subject of major concern. However, the study of these death phenomenons is hampered by the fact that the environment of foam cells is particularly complex and difficult to reproduce in vitro. Indeed, these cells are exposed to a variety of oxidized/modified lipoproteins, subject to massive cytokine stimulation and can be exposed to low oxygen tension. This latest, also called hypoxia, is an environmental condition well known to modulate different cellular mechanisms such as lipid storage, inflammation and death processes. If the effects of hypoxia on tumor cells are well studied, the effects of hypoxia on foam cells in atherosclerosis are still largely unknown. The aim of our work is to compare the effects of two different types of oxidized LDL on lipid load, on unfolded protein response (UPR) and on death processes in macrophages-derived foam cells as well as to study the effects of hypoxia on these mechanisms. In this work we used two different types of oxidized/modified LDL to generate foam cells out of THP-1 derived macrophages. As the physiological relevance of the traditional copper sulphate oxidized LDL is still a matter of debate it is of interest to settle new experimental models. Hence, in parallel to copper sulphate oxidized LDL or Ox-LDL, we used LDL oxidized with myeloperoxidase (MPO), a peroxidase found to be present and active in atheroma. In contrast to Ox-LDL which are oxidized both on lipidic and proteic moieties, the LDL oxidized by MPO called Mox-LDL, are only modified on apoB. In the first part, we established the experimental conditions giving rise to a marked foam cell phenotype with both types of LDL. We determined that a 72 hours incubation in the presence of 100 µg/ml with Ox-LDL or Mox-LDL led to heavily loaded cells. The cells incubated in the presence of Mox-LDL displayed a larger lipid load than the cells incubated with the same concentration of Ox-LDL for the same period of time. Then, we studied the effect of hypoxia on lipid load of cell incubated with Ox-LDL or Mox-LDL. The incubation of macrophages with Ox-LDL or Mox-LDL under hypoxia (1 % O2) did not influence the lipid load. In the second part, stress-induced pathways were investigated. Both Ox-LDL and Mox-LDL triggered the UPR, but in a different way. While the cells incubated for 72 hours in the presence of Ox-LDL displayed pronounced UPR with marked eIF2α phosphorylation and pronounced XBP-1 splicing, the cells incubated for 72 hours in the presence of Mox-LDL presented low level eIF2α phosphorylation and moderate XBP-1 splicing. The cells incubated with these two types of LDL under hypoxia showed less pronounced markers of UPR, suggesting a protective effect of this environmental condition. We also showed that both types of LDL were cytotoxic after 72 hours of incubation, the Mox-LDL to a larger extent than the Ox-LDL. Cells incubated in the presence of Ox-LDL and to a larger extent with Mox-LDL, showed markers of apoptosis (PARP cleavage, α-fodrin cleavage, caspases 3 and 7 cleavage). With both types of LDL, the amount of apoptotic markers was decreased when the cells were incubated under hypoxia, again suggesting a protective effect of low oxygen tension. As we evidenced that hypoxia induced the overexpression of different chaperones, such as Grp78, Grp94 and ERO1, we hypothesized that the protection provided by hypoxia partly depended on these proteins. The autophagy which is described to be triggered by ER stress such as UPR was finally studied. We evidenced that the cells incubated with both types of oxidized LDL displayed increased features of macroautophagy, without influence of hypoxia. We hypothesized that even if Ox-LDL and Mox-LDL triggered autophagy, the purpose of the process is different for each type of LDL. While autophagy would be protective and would prevent apoptosis in the cells incubated in the presence of Ox-LDL, it may be lethal and act in synergy with apoptosis to induce cell death in the cells incubated in the presence of Mox-LDL. Altogether, these results show that foam cells can be obtained with macrophage incubation with different types of oxidized LDL. However, the foam cells obtained with the different oxidized LDL displayed different stress associated signaling pathways. To unravel these different signaling pathways as well as to understand the effects of hypoxia on these signaling pathways in foam cell is important to design and improve future therapeutic strategies.
Etude des voies de réponse aux stress dans des macrophages humains différenciés en cellules spumeuses en présence de différents types de LDL, en hypoxie et en normoxie
Vlaminck, B. (Author). 3 Sept 2012
Student thesis: Doc types › Doctor of Sciences