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Identification of cholesterol crystals in plaques of atherosclerotic mice using hyperspectral CARS imaging

Abstract
The accumulation of lipids within the arterial wall plays a critical role in the progression of atherosclerosis. As monocytes are recruited to the vessel wall during the initial inflammatory response, they differentiate into macrophage cells and begin to ingest oxidized low density lipoproteins (LDL), thus forming specialized foam cells. The LDL particles, which are rich in cholesterol, are unable to be processed by the macrophages and therefore cause the foam cells to eventually rupture. The death of these foam cells further propagates the inflammatory process, recruiting more monocyte-derived macrophages and consequently leaving behind a growing pool of cholesterol and other lipophilic compounds. Cholesterol, in the form of cholesterol crystals (ChC), plays a particularly prominent role in the atherogenic process and serves as a marker for advanced atherosclerotic lesions. Studies have shown that these crystals exist within sites of plaque rupture, but they are typically absent from deceased patients who suffered from severe atherosclerosis (but not as their primary cause of death), thereby suggesting an important link between ChC formation and atherosclerosis-related death (6, 10, 11). Recently, ChCs have been recognized as capable of inducing an inflammatory response through stimulation of the caspase-1-activating NLRP3 inflammasome. In addition, the formation of extracellular ChC material has been implicated as a mechanical factor that contributes directly to plaque vulnerability.

Although several advances have been made in the detection and imaging of these lipid structures in plaque lesions, their morphology and composition have yet to be fully elucidated, particularly in different animal models of disease. To address this issue, we analyzed lipid morphology and composition in the atherosclerotic plaques of two animal models of disease, the low density lipoprotein receptor-deficient (LDLR(-/-)) mouse and the ApoE lipoprotein-deficient (ApoE(-/-)) mouse, utilizing hyperspectral coherent anti-Stokes Raman scattering (CARS) microscopy in combination with principal component analysis (PCA) (LAMMP TRD-1). Hyperspectral CARS imaging revealed lipid-rich macrophage cells and condensed needle-shaped and plate-shaped lipid crystal structures in both mice. Spectral analysis with PCA and comparison to spectra of pure cholesterol and cholesteryl ester derivatives further revealed these lipid structures to be pure cholesterol crystals, which were predominantly observed in the ApoE(-/-) mouse model. These results illustrate the ability of hyperspectral CARS imaging in combination with multivariate analysis to characterize atherosclerotic lipid morphology and composition with chemical specificity, and consequently, provide new insight into the formation of cholesterol crystal structures in atherosclerotic plaque lesions.

Impact
We have established that hyperspectral CARS imaging is capable of chemically identifying cholesterol crystals and discriminating them from other condensed aliphatic lipid microstructures in atherosclerotic plaques. While we have found cholesterol crystal aggregation throughout the aorta of 24-week-old ApoE./. mice, the distribution of crystals in LDLR./. mice of similar age is less abundant, thereby suggesting a possible discrepancy in plaque lesion development between the two atherosclerotic models. These findings elucidate several key aspects of the atherosclerotic mouse and provide a foundation for future studies on the role of cholesterol crystals during atherosclerotic disease progression.

Citation
Lim RS, Suhalim JL, Miyazaki-Anzai S, Miyazaki M, Levi M, Potma EO, Tromberg BJ. Identification of cholesterol crystals in plaques of atherosclerotic mice using hyperspectral CARS imaging. J Lipid Res. 2011 Dec;52(12):2177-86. Epub 2011 Sep 23. PMC3220286

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