The regional ratio of cholesteryl palmitate to cholesteryl oleate measured by ToF-SIMS as a key parameter of atherosclerosis.

Changes in cholesterol ester (CE) content regulate the progression of atherosclerosis. However, the spatial dynamics of CE subsets and their quantitative changes during lesion progression are not well understood due to a lack of appropriate imaging techniques. In this study, we developed an imaging-based analysis method to map the distribution of CE subsets using time-of-flight secondary ion mass spectrometry (ToF-SIMS). Serial sections of atherosclerotic aortic sinuses from apolipoprotein E knock-out mice (n = 15) fed a 0.15% high-fat diet for 12-20 weeks were examined by ToF-SIMS. We found that the ratio of cholesteryl palmitate (Ch-PA) to cholesteryl oleate (Ch-OA) increased by approximately 99% (p = 0.02) as atherosclerosis progressed, whereas the ratios of cholesteryl linoleate (p = 0.09) and cholesteryl stearate (p = 0.22) to Ch-OA did not change significantly. In advanced atherosclerotic plaques, in situ and in-vitro cell death assays showed that local Ch-PA densities were highly correlated with an increase in the number of apoptotic cells. These results suggest that increased Ch-PA may contribute to the formation of a necrotic core by increasing cell death. Our results indicate that the regional ratio of CEs as measured by ToF-SIMS might be a valuable new marker of atherosclerotic progression.