Identification and visualization of oxidized lipids in atherosclerotic plaques by microscopic imaging mass spectrometry-based metabolomics

Background and aims: Dysregulated lipid metabolism has emerged as one of the major risk factors of atherosclerosis. Presently, there is a consensus that oxidized LDL (oxLDL) promotes development of atherosclerosis and downstream chronic inflammatory responses. Due to the dynamic metabolic disposition of lipoprotein, conventional approach to purify bioactive lipids for subsequent comprehensive analysis has proven to be inadequate for elucidation of the oxidized lipids species accountable for pathophysiology of atherosclerotic lesions. Herein, we aimed to utilize a novel mass microscopic imaging technology, coupled with mass spectrometry (MS) to characterize oxidized lipids in atherosclerotic lesions. Methods: We attempted to use MALDI-TOF-MS and iMScope to identify selected oxidized lipid targets and visualize their respective localizations in study models of atherosclerosis. Results: Based on the MS analysis, detection of 7-K under positive ionization through product ion peak at m/z 383 [M + H–H₂O] indicated the distinctive presence of targeted lipid within Cu²⁺-oxLDL and Cu²⁺-oxLDL loaded macrophage-like J774A.1 cells, along with other cholesterol oxidation products. Moreover, the application of two-dimensional iMScope has successfully visualized the localization of lipids in aortic atherosclerotic plaques of the Watanabe heritable hyperlipidemic (WHHL) rabbit. Distinctive lipid distribution profiles were observed in atherosclerotic lesions of different sizes, especially the localizations of lysoPCs in atherosclerotic plaques. Conclusions: Taken together, we believe that both MALDI-TOF-MS and iMScope metabolomics technology may offer a novel proposition for future pathophysiological studies of lipid metabolism in atherosclerosis.