A New Imaging Technique for Characterization of Cholesterol Crystal Growth in Atherosclerotic Cell Models

Background: Accumulation of cholesterol in artery walls is a prominent feature of atherosclerosis, a major precursor of most cardiovascular diseases. The irreversible formation of cholesterol crystals is associated with increased vulnerability of the plaque. Herein we address the question of how cholesterol crystals form in atherosclerosis. Using macrophage cell models, we examine whether cholesterol precipition initiate from cell membranes following LDL processing.

Methods: macrophage cells were incubated with acLDL particles for different time lengths and the cholesterol segregation process was followed, using an antibody that identifies the newly formed cholesterol crystals. Direct imaging was achieved using a recently developed correlative method that combines cryo soft X-ray tomography with stochastic optical reconstruction microscopy (STORM). This is a novel technique that can potentially be applied to other biological and pathological systems.

Results: At early incubation stages we identified small rhomb-like crystals (200−400 nm) closely associated with cell plasma membranes. When cells are incubated with acLDL for longer time periods, large rod-like crystals (~10 µm) grow at intracellular locations. Using cryo- electron diffraction, we identified that the large crystals correspond to a different polymorphic structure of cholesterol. The cell membrane environment determines the crystal form.

Conclusion: Our findings show that different crystals form at different cellular locations. We believe that our findings, besides explaining the different crystal morphologies found in human plaques, may contribute to elucidating the mechanisms by which cholesterol pathologically crystallizes in cells.