Two-Photon Excited Fluorescence (TPEF) may be useful to identify macrophage subsets based on their metabolic activity and cellular responses in atherosclerotic plaques

Abstract Background and aims Atherosclerosis is characterized by the formation of lipid plaques within the arterial wall. In such plaques, the massive and continuous recruitment of circulating monocyte-derived macrophages induces inflammation, leading to plaque destabilization and rupture. Plaque vulnerability is linked to the presence of (i) a large lipid core that contains necrotic, “foamy” macrophages (FMs), (ii) a thin fibrous cap that cannot limit the prothrombotic lipid core, and potentially (iii) an imbalance between inflammatory and immunoregulatory macrophages. These opposite macrophage functions rely on the use of different energy pathways (glycolysis and oxidative phosphorylation, respectively) that may lead to different levels of the auto-fluorescent cofactors nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD). We hypothesized that high-resolution two-photon excited autofluorescence (TPEF) imaging of these cofactors may be used to monitor the metabolic activity and cellular responses of macrophages in atherosclerotic plaques. Methods Different models of human FMs were generated by exposure to acetylated or oxidized low-density lipoproteins (LDL), with/without human carotid extract (CE). Their phenotype and optical properties were compared with those of extremely polarized macrophages, inflammatory M1 (MLPS+IFNγ) and immunoregulatory M2 (MIL4+IL13). Results These FM models displayed an intermediate phenotype with low levels of M1 and M2 “specific” markers. Moreover, the NADH and FAD autofluorescence profiles of FMoxLDL+/-CE cells were significantly distinct from those of M1 and M2 macrophages. Conclusions TPEF imaging may be useful to follow the metabolic activity and cellular responses of the different macrophage subtypes present in atherosclerotic plaques in order to detect vulnerable areas.