L’athérosclérose est un processus d’évolution lente, dont les premières étapes sont marquées par le dépôt de lipoprotéines de basse densité (LDL) dans l’intima des artères. Les études histologiques montrent que les cellules musculaires lisses (CML) sont prépondérantes dans l’intima des lésions précoces et qu’elles ont un rôle critique dans le développement des lésions. A l’inverse des CML saines de la média qui présentent un phénotype contractile, certaines CML de l'intima sont caractérisées par la synthèse de matrice extracellulaire, associée à une migration et une prolifération augmentées. Comme les macrophages, une fraction de ces CML peut internaliser les LDL et participe à la formation de cellules spumeuses.Dans ce travail, nous avons cherché à déterminer le rôle du syndécan-1 dans la modulation du phénotype de la CML. Nous avons mis en évidence une surexpression du syndécan-1 par les CML intimales lors des stades précoces de l'athérosclérose. Dans le but de déterminer l'effet de cette surexpression, nous avons élaboré un vecteur lentiviral codant le syndécan-1 afin de le surexprimer dans les CML en culture. Nos résultats montrent qu'en réponse au transforming growth factor beta (TGF-β), les CML surexprimant le syndécan-1 présentent une augmentation amplifiée des gènes du collagène de type I et de certains marqueurs de fonction contractile incluant la SM α-actine, la calponine et la smootheline.Un autre volet de l'étude s'est intéressé à l'origine des cristaux de cholestérol (CCs) dans les lésions. Fréquemment retrouvés dans les lésions avancées, les études montrent que ces CCs pourraient induire mécaniquement la rupture de la plaque. Par l'analyse de coupes d'aortes fraîches, nous avons observé que les CCs apparaissent au stade de la lésion fibrolipidique et qu'ils sont associés à la présence de CML mortes. In vitro, les CML chargées en cholestérol sont capables de produire des cristaux, un processus qui est accéléré par la présence de collagène de type I et par l'inhibition de l'autophagie et de l'estérification du cholestérol. Nous avons montré que le collagène de type I entraine une diminution de p62 et une augmentation de Niemann-Pick C1, suggérant qu'il module l'expression de gènes associés à l'autophagie et au trafic du cholestérol.L’ensemble de ce travail ouvre ainsi de nouvelles perspectives quant à l’identification des mécanismes initiant la modulation du phénotype de CML, et aux conséquences de cette modulation au niveau de la lésion. / Atherosclerosis is a chronic pathological process that is characterized, in the earliest stage, by low density lipoproteins (LDL) accumulation within the arterial intima. Evidences show that smooth muscle cells (SMCs) are preponderant in the intima of early lesions and play a major role in lesions development. Whereas medial SMCs are involved in the contractile function, intimal SMCs are characterized by increased proliferation and migration, loss of contractile markers, and extracellular matrix production. Like macrophages, these SMCs have the capacity to internalize LDL and become foam cells. In this work, we explored syndecan-1 role in SMCs phenotypic modulation. We show that syndecan-1 expression is increased in intimal SMCs of early lesions. To determine the effect of this overexpression in SMCs in vitro, we elaborated the construction of a lentiviral vector encoding syndecan-1 cDNA. Our results demonstrate that syndecan-1 amplifies the up-regulation of type I collagen, SM α-actin, calponin, and smoothelin mRNA expression, induced by TGF-β.In the second part of our work, we investigated cholesterol crystals (CCs) formation in atherosclerotic lesions. Besides to being a major component of the atheromatous core, studies have shown that CCs could promote plaque rupture by causing mechanical damage. Our observations of fresh aortas revealed that CCs first appeared at the fibroatheroma transition and are associated with the death of SMC. Cholesterol-loaded human SMCs were capable of producing CCs in vitro, a process that was enhanced by type I collagen and by inhibition of autophagy and cholesterol esterification. We found that type I collagen leads to a decrease in the expression of p62, and an increase in Niemann-Pick C1, suggesting that collagen induces changes in genes relating to regulation of intracellular cholesterol levels and localization.In conclusion, this work described a novel candidate potentially implicated in SMCs phenotypic modulation, and the important consequences of this modulation in lesions development.

Cholesterol crystals have been linked to activation of the NLRP3 inflammasome and the formation of foreign body giant cells (FBGCs). It has been hypothesized that FBGCs have a role in advanced atherosclerotic plaque formation. This thesis examined the feasibility of producing stable cultures of FBGCs starting with human monocytes with the goal to examine pterin production by these cells in comparison to human monocyte derived macrophages (HMDMs). The study also investigated the effect of cholesterol crystals on 7,8-dihydroneopterin (7,8-NP) production and modulation of IL-1β levels in macrophages. 7,8-Dihydroneopterin is a potent antioxidant generated by macrophages which also down regulates the expression of macrophage scavenger receptor CD36. The use of alpha-tocopherol and IL-4 as FBGC fusion mediators was explored. Using these mediators, large numbers of FBGC were successfully cultured. The rates of fusion achieved in the cultures were low, and the cells had poor adhesion, which prevented pterin measurement. FBGC, which are thought to remove crystallized cholesterol from the plaque, cleared 21% of cholesterol crystal compared to 50% cleared by HMDM cells. Due to this result, the effect of cholesterol crystals on pterin production in monocytes and macrophages was explored. Cholesterol crystals cause inflammation through the activation of the NLRP3 inflammasome, however, it was unknown whether they could modulate 7,8-NP production. Cholesterol crystals caused an intracellular dose-dependent loss of 7,8-NP to its oxidized form, neopterin, in HMDM cells. Cholesterol crystals induced intracellular synthesis of 7,8-NP in HMDMs. 7,8-NP was released into the supernatant and oxidized to neopterin in media. Monocytes treated with cholesterol crystals released up to 100 nM of neopterin and 120 nM of 7,8-NP in the media after 48 hours. The combination of IFN- and cholesterol crystals appeared to inhibit the release of 7,8-NP into the media for the first 48 hours, after this time 7,8-NP release rapidly increased. The addition of exogenous 200 μM 7,8-NP showed that in the presence of monocytes, cholesterol crystals did not cause the oxidation of 7,8-NP to neopterin, as seen in HMDMs but possibly to 7,8-dihydroxanthopterin or xanthopterin. The presence of 7,8-NP increased IL-1β expression in the presence of cholesterol crystals after 24 hours incubation. FBGCs and the removal of cholesterol crystals may be a key process in the resolution of atherosclerotic plaques. It appears that cholesterol crystals are able to modulate inflammatory processes including activation of the inflammasome and balance of 7,8-dihydroneopterin to the oxidized neopterin. The infiltrating monocytes may provide antioxidant protection against the inflammation induced by cholesterol crystals and the activity of the infammasome.