Background: Atherosclerosis is a progressive disease characterized by the sub-endothelial accumulation of Low-Density Lipoproteins (LDL), cholesterol-esters and cholesterol crystals. Cholesterol crystals facilitate the instability and rupture of the atheromatous plaque, leading to arterial thrombosis. Macrophage cells have a crucial role in the process of cholesterol crystal formation. This research aims to elucidate cholesterol crystal formation in atherosclerotic lesions in 3D and at high resolution.
Methods: J774A.1 macrophages were incubated with Acetylated-LDL, viability and cholesterol deposition were monitored using trypan blue and an antibody specific to cholesterol crystals. Cryo-Scanning Electron Microscopy (cryo-SEM) combined with Cathode-Luminescence (CL) was employed for the identification and localization of cholesterol crystals within macrophages/atherosclerotic tissues. For 3D analysis of macrophage foam cells/plaques, we employed cryo-Focused Ion Beam/Scanning (cryo-FIB-SEM) block face serial imaging.
Results: We developed a correlative technique combining cryo-SEM with CL for the inspection of cholesterol crystals found within macrophage cells and atheromatous plaque sections. Large amounts of rod-like cholesterol crystals within Ac-LDL fed macrophages and stacks of plate-like cholesterol crystals in different regions of atherosclerotic lesions were observed with high spatial resolution. We found that crystals are formed within viable J774A.1, implying that the crystallization process may occur during the initial stages of LDL accumulation and is not necessarily associated with macrophage cell death.
Conclusions: High-resolution 3D imaging of atheromatous tissues and cells provides new insight into pathological cholesterol crystal formation and processing. Cholesterol crystal formation is a process associated with early atherogenic events.