3D cryoimaging of cell-mediated cholesterol crystal clearance in human atherosclerotic lesions.

We applied micro-computed tomography, high-resolution cryo-scanning electron microscopy (SEM) combined with cathodoluminescence, and cryo-focused ion beam Milling-SEM to perform three-dimensional imaging of human atherosclerotic tissues with tens of nanometers resolution, under hydrated, near-native conditions with minimal sample processing. The same technology was applied to cultured macrophages exposed to cholesterol crystals, and the observations made on the macrophages were compared to those made on the pathological tissue. We observed that cholesterol crystal digestion and, eventually, cholesterol crystal clearance occurs in the advanced human plaques through cellular processing.

Examining atherosclerotic lesions in three dimensions at the nanometer scale with cryo-FIB-SEM.

We employed in a correlative manner an unconventional combination of methods, comprising cathodoluminescence, cryo-scanning electron microscopy (SEM), and cryo-focused ion beam (FIB)-SEM, to examine the volumes of thousands of cubed micrometers from rabbit atherosclerotic tissues, maintained in close-to-native conditions, with a resolution of tens of nanometers. Data from three different intralesional regions, at the media-lesion interface, in the core, and toward the lumen, were analyzed following segmentation and volume or surface representation. The media-lesion interface region is rich in cells and lipid droplets, whereas the core region is markedly richer in crystals and has lower cell density.

Structural analysis of lecithin:cholesterol acyltransferase bound to high density lipoprotein particles.

Lecithin:cholesterol acyltransferase (LCAT) catalyzes a critical step of reverse cholesterol transport by esterifying cholesterol in high density lipoprotein (HDL) particles. LCAT is activated by apolipoprotein A-I (ApoA-I), which forms a double belt around HDL, however the manner in which LCAT engages its lipidic substrates and ApoA-I in HDL is poorly understood. Here, we used negative stain electron microscopy, crosslinking, and hydrogen-deuterium exchange studies to refine the molecular details of the LCAT-HDL complex.