The importance of macrophage polarization through atherogenesis is established. However, most studies rely on immunohistological approaches, which have several limitations, such as precluding comprehensive phenotypic analysis. The aim of this study was to perform an alternative analysis of macrophage phenotypes in advanced human atherosclerotic plaques and compare them with their presence in non-atherosclerotic arteries. Atherosclerotic plaques from 70 individuals indicated for carotid endarterectomy, and samples of non-atherosclerotic arterial tissue (renal artery, control group) from 45 living kidney donors were processed to obtain immunocytes and incubated with antibodies (CD45, CD14, CD16, CD36, CD163, and CD206) to be analyzed by flow cytometry. Macrophages in the atherosclerotic plaques tend to express CD16 more intensively than in non-atherosclerotic arterial tissue (transient, CD16low p < 0.001, pro-inflammatory, CD16high p < 0.001), and the expression is more closely associated with CD36 expression. Both transient and pro-inflammatory macrophages are linked with the CD206-CD163+ or CD206+CD163+ phenotype in atherosclerotic plaques, while CD206-CD163- dominates within the anti-inflammatory (CD16neg) population in the control group. Interestingly, when evaluating all macrophages (regardless of CD16 expression), almost all are CD163+ in both groups, supporting the critical importance of using a combination of specific markers. Our results provide a deeper insight into macrophage subpopulations in advanced human atherosclerotic plaques compared with those in non-atherosclerotic vessels. Additionally, our data highlight the critical importance of using appropriate techniques, such as flow cytometry, allowing for simultaneous analysis of multiple markers to accurately and comprehensively characterize macrophages within the atherosclerotic plaque.

Department of Anaesthesia and Intensive Medicine 1st Faculty of Medicine Charles University and Military University Hospital Prague Czech Republic

Department of Cardiology Institute for Clinical and Experimental Medicine Prague Czech Republic

Department of Epidemiology and Biostatistics 3rd Faculty of Medicine Charles University Prague Czech Republic

Department of Pathology Institute for Clinical and Experimental Medicine Prague Czech Republic

Department of Physiology Faculty of Science Charles University Prague Czech Republic

Department of Transplant Surgery Institute for Clinical and Experimental Medicine Prague Czech Republic

Laboratory for Atherosclerosis Research Centre for Experimental Medicine Institute for Clinical and Experimental Medicine Prague Czech Republic

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