Oxysterols Are Not Functionally Equivalent: Cell-Type- and Territory-Specific Responses in Human Models of Atherosclerosis

This study aimed to compare the biological activities of major oxysterols, including 7-ketocholesterol (7KC), 7α-hydroxycholesterol (7α-HC), 7β-hydroxycholesterol (7β-HC), and 27-hydroxycholesterol (27HC), as well as oxidized LDL (oxLDL), in complementary human models representative of the atherosclerotic microenvironment: monocyte-derived macrophages (MΦs), vascular endothelial cells (v-ECs), and vascular smooth muscle cells (v-SMCs) isolated from the carotid artery and abdominal aorta. Cellular responses were strongly dependent on both cell type and vascular territory. Among the oxysterols tested, 7KC exerted the strongest biological effects, markedly reducing cell viability, increasing oxidative stress and apoptosis, promoting endothelial dysfunction, and profoundly altering lipid homeostasis pathways. Spectral Nile Red analysis further revealed distinct patterns of neutral-lipid- and phospholipid-associated fluorescence, indicating oxysterol-specific remodeling of intracellular lipid compartments. In vascular smooth muscle cells, 7KC was associated with increased CD36 expression and reduced ABCA1/ABCG1-mediated cholesterol efflux, whereas macrophages responded predominantly through SREBP1- and NR1H4-associated metabolic reprogramming rather than enhanced lipid uptake. In contrast, oxLDL was the strongest inducer of neutral lipid accumulation and classical foam-cell formation in macrophages. Although 27HC increased intracellular 7KC levels, it induced comparatively limited effects on viability, oxidative stress, and macrophage functions. Moreover, oxLDL promoted a distinct macrophage activation state and altered polarization trajectories depending on the timing of exposure. These findings demonstrate that major oxysterols exert distinct and complementary biological effects across vascular and immune cell populations, highlighting their differential contribution to atherosclerotic plaque development and progression, and support the development of therapeutic approaches targeting multiple oxysterol species rather than a single oxidized cholesterol derivative.