Autophagy has been shown to be stimulated in advanced atherosclerotic plaques by metabolic stress, inflammation and oxidized lipids. The lack of published studies addressing the potential stimulation of pro-survival autophagy by oxysterols, a family of cholesterol oxidation products, has prompted our study. Thus, the goal of the current study is to elucidate the molecular mechanism of the autophagy induced by 27-hydroxycholesterol (27-OH), that is one of the most abundant oxysterols in advanced atherosclerotic lesions, and to assess whether the pro-oxidant effect of the oxysterol is involved in the given response. Here we showed that 27-OH, in a low micromolar range, activates a pro-survival autophagic response in terms of increased LC3 II/LC3 I ratio and Beclin 1, that depends on the up-regulation of extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K)/Akt pathways as a potential result of an intracellular reactive oxygen species increase provoked by the oxysterol in human promonocytic U937 cells. Moreover, 27-OH induced autophagy is dependent on the relation between nuclear factor erythroid 2 p45-related factor 2 (Nrf2)-dependent antioxidant response and p62. The data obtained highlight the involvement of cholesterol oxidation products in the pathogenesis of oxidative stress related chronic diseases like atherosclerosis. Therefore, deeply understanding the complex mechanism and generating synthetic or natural molecules targeting this survival mechanism might be very promising tools in the prevention of such diseases.

The hypercholesterolemia-atherosclerosis association is now established; hypercholesterolemia may induce vascular-cell activation, subsequently increasing expression of adhesion molecules, cytokines, chemokines, growth factors, and other key inflammatory molecules. Among inflammatory molecules expressed by vascular cells, integrins play a critical role in regulating macrophage activation and migration to the site of inflammation, by mediating cell-cell and cell-extracellular matrix interactions. The main lipid oxidation products present in oxidized LDL that may be responsible for inflammatory processes in atherogenesis, are cholesterol oxidation products, known as oxysterols. This study demonstrates the effect of an oxysterol mixture, compatible with that detectable in human hypercholesterolemic plasma, on the expression and synthesis of β₁-integrin in cells of the macrophage lineage. The molecular signaling whereby oxysterols induce β₁-integrin up-regulation is also comprehensively investigated. Over-expression of β₁-integrin depends on activation of classic and novel members of protein kinase C and extracellular signal-regulated kinases 1 and 2, as well as of the up-stream G-protein (Gq and G13), c-Src, and phospholipase C. In addition, the localization of β₁-integrin in advanced human carotid plaques is highlighted, marking its importance in atherosclerotic plaque progression.