Atherosclerosis is a chronic inflammatory disease of the blood vessels caused by elevated levels of lipoproteins. The hyperlipoproteinemia triggers a series of cellular changes, particularly the activation of the macrophages, which play a crucial role in the development and progression of atherosclerosis. The presence of free cholesterol (FC) in lipoproteins may contribute to macrophage stimulation. However, the mechanisms linking the accumulation of FC in macrophages to their pro-inflammatory activation remain poorly understood. Our research found a positive correlation between the number of pro-inflammatory macrophages (CD14 + CD16 + CD36high) in visceral adipose tissue and the levels of LDL-C and cholesterol remnant particles in 56 healthy people. In contrast, the proportion of anti-inflammatory, alternatively activated macrophages (CD14 + CD16-CD163+) correlated negatively with HDL-C. Additionally, our in vitro study demonstrated that macrophages accumulating FC promoted a pro-inflammatory response, activating the TNF-α and chemokine CCL3 genes. Furthermore, the accumulation of FC in macrophages alters the surface receptors on macrophages (CD206 and CD16) and increases cellular granularity. Notably, the CD36 surface receptor and the ACAT and CD36 genes did not show a response. These results suggest a link between excessive FC accumulation and systemic inflammation to underlie the development of atherosclerosis.

Department of Data Science Institute for Clinical and Experimental Medicine Prague Czech Republic

Department of Physiology Faculty of Science Charles University Prague Czech Republic

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

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

See more in PubMed

Ambarus, C. A. , Krausz, S. , van Eijk, M. , Hamann, J. , Radstake, T. R. D. J. , Reedquist, K. A. , Tak, P. P. , & Baeten, D. L. P. (2012). Systematic validation of specific phenotypic markers for in vitro polarized human macrophages. Journal of Immunological Methods, 375, 196–206. PubMed

Bagheri, B. , Alikhani, A. , Mokhtari, H. , & Rasouli, M. (2018). The ratio of Unesterified/esterified cholesterol is the major determinant of Atherogenicity of lipoprotein fractions. Medical Archives, 72, 103–107. PubMed PMC

Brewer, H. B., Jr. (2011). Clinical review: The evolving role of HDL in the treatment of high‐risk patients with cardiovascular disease. The Journal of Clinical Endocrinology and Metabolism, 96, 1246–1257. PubMed

Buwaneka, P. , Ralko, A. , Liu, S.‐L. , & Cho, W. (2021). Evaluation of the available cholesterol concentration in the inner leaflet of the plasma membrane of mammalian cells. Journal of Lipid Research, 62, 100084. PubMed PMC

Castañer, O. , Pintó, X. , Subirana, I. , Amor, A. J. , Ros, E. , Hernáez, Á. , Martínez‐González, M. Á. , Corella, D. , Salas‐Salvadó, J. , Estruch, R. , Lapetra, J. , Gómez‐Gracia, E. , Alonso‐Gomez, A. M. , Fiol, M. , Serra‐Majem, L. , Corbella, E. , Benaiges, D. , Sorli, J. V. , Ruiz‐Canela, M. , … Fitó, M. (2020). Remnant cholesterol, not LDL cholesterol, is associated with incident cardiovascular disease. Journal of the American College of Cardiology, 76, 2712–2724. PubMed

Čejková, S. , Králová Lesná, I. , Froněk, J. , Janoušek, L. , Králová, A. , Ždychová, J. , & Poledne, R. (2017). Pro‐inflammatory gene expression in adipose tissue of patients with atherosclerosis. Physiological Research, 66, 633–640. PubMed

Chinetti‐Gbaguidi, G. , Baron, M. , Bouhlel, M. A. , Vanhoutte, J. , Copin, C. , Sebti, Y. , Derudas, B. , Mayi, T. , Bories, G. , Tailleux, A. , Haulon, S. , Zawadzki, C. , Jude, B. , & Staels, B. (2011). Human atherosclerotic plaque alternative macrophages display low cholesterol handling but high phagocytosis because of distinct activities of the PPARγ and LXRα pathways. Circulation Research, 108, 985–995. PubMed PMC

Christian, A. E. , Haynes, M. P. , Phillips, M. C. , & Rothblat, G. H. (1997). Use of cyclodextrins for manipulating cellular cholesterol content. Journal of Lipid Research, 38, 2264–2272. PubMed

Coen, P. M. , Flynn, M. G. , Markofski, M. M. , Pence, B. D. , & Hannemann, R. E. (2010). Adding exercise to rosuvastatin treatment: Influence on C‐reactive protein, monocyte toll‐like receptor 4 expression, and inflammatory monocyte (CD14+CD16+) population. Metabolism, 59, 1775–1783. PubMed

Cordero, A. , Alvarez‐Alvarez, B. , Escribano, D. , García‐Acuña, J. M. , Cid‐Alvarez, B. , Rodríguez‐Mañero, M. , Quintanilla, M. A. , Agra‐Bermejo, R. , Zuazola, P. , & González‐Juanatey, J. R. (2023). Remnant cholesterol in patients admitted for acute coronary syndromes. European Journal of Preventive Cardiology, 30, 340–348. PubMed

Das, A. , Brown, M. S. , Anderson, D. D. , Goldstein, J. L. , & Radhakrishnan, A. (2014). Three pools of plasma membrane cholesterol and their relation to cholesterol homeostasis. eLife, 3, e02882. PubMed PMC

Feng, M. , Darabi, M. , Tubeuf, E. , Canicio, A. , Lhomme, M. , Frisdal, E. , Lanfranchi‐Lebreton, S. , Matheron, L. , Rached, F. , Ponnaiah, M. , Serrano, C. V., Jr. , Santos, R. D. , Brites, F. , Bolbach, G. , Gautier, E. , Huby, T. , Carrie, A. , Bruckert, E. , Guerin, M. , … Kontush, A. (2020). Free cholesterol transfer to high‐density lipoprotein (HDL) upon triglyceride lipolysis underlies the U‐shape relationship between HDL‐cholesterol and cardiovascular disease. European Journal of Preventive Cardiology, 27, 1606–1616. PubMed

Ferrari, A. , He, C. , Kennelly, J. P. , Sandhu, J. , Xiao, X. , Chi, X. , Jiang, H. , Young, S. G. , & Tontonoz, P. (2020). Aster proteins regulate the accessible cholesterol Pool in the plasma membrane. Molecular and Cellular Biology, 40, e00255. PubMed PMC

Gerl, M. J. , Vaz, W. L. C. , Domingues, N. , Klose, C. , Surma, M. A. , Sampaio, J. L. , Almeida, M. S. , Rodrigues, G. , Araújo‐Gonçalves, P. , Ferreira, J. , Borbinha, C. , Marto, J. P. , Viana‐Baptista, M. , Simons, K. , & Vieira, O. V. (2018). Cholesterol is inefficiently converted to cholesteryl esters in the blood of cardiovascular disease patients. Scientific Reports, 8, 14764. PubMed PMC

Gillard, B. K. , Rosales, C. , Gotto, A. M. J. , & Pownall, H. J. (2023). The pathophysiology of excess plasma‐free cholesterol. Current Opinion in Lipidology, 34, 278–286. PubMed PMC

Goldenthal, K. L. , Pastan, I. , & Willingham, M. C. (1984). Initial steps in receptor‐mediated endocytosis. The influence of temperature on the shape and distribution of plasma membrane clathrin‐coated pits in cultured mammalian cells. Experimental Cell Research, 152, 558–564. PubMed

Goldstein, J. L. , & Brown, M. S. (1973). Familial hypercholesterolemia: Identification of a defect in the regulation of 3‐hydroxy‐3‐methylglutaryl coenzyme a reductase activity associated with overproduction of cholesterol. Proceedings of the National Academy of Sciences of the United States of America, 70, 2804–2808. PubMed PMC

Goldstein, J. L. , & Brown, M. S. (2001). Molecular medicine. The cholesterol quartet. Science, 292, 1310–1312. PubMed

Grosheva, I. , Haka, A. S. , Qin, C. , Pierini, L. M. , & Maxfield, F. R. (2009). Aggregated LDL in contact with macrophages induces local increases in free cholesterol levels that regulate local actin polymerization. Arteriosclerosis, Thrombosis, and Vascular Biology, 29, 1615–1621. PubMed PMC

Hjuler Nielsen, M. , Irvine, H. , Vedel, S. , Raungaard, B. , Beck‐Nielsen, H. , & Handberg, A. (2015). Elevated atherosclerosis‐related gene expression, monocyte activation and microparticle‐release are related to increased lipoprotein‐associated oxidative stress in familial hypercholesterolemia. PLoS One, 10, e0121516. PubMed PMC

Jakulj, L. , v. Dijk, T. H. , d. Boer, J. F. , Kootte, R. S. , Schonewille, M. , Paalvast, Y. , Boer, T. , Bloks, V. W. , Boverhof, R. , Nieuwdorp, M. , Beuers, U. H. W. , Stroes, E. S. G. , & Groen, A. K. (2016). Transintestinal cholesterol transport is active in mice and humans and controls ezetimibe‐induced fecal neutral sterol excretion. Cell Metabolism, 24(6), 783–794. 10.1016/j.cmet.2016.10.001 PubMed DOI

Jepsen, A.‐M. K. , Langsted, A. , Varbo, A. , Bang, L. E. , Kamstrup, P. R. , & Nordestgaard, B. G. (2016). Increased remnant cholesterol explains part of residual risk of all‐cause mortality in 5414 patients with ischemic heart disease. Clinical Chemistry, 62, 593–604. PubMed

Kralova Lesna, I. , Poledne, R. , Fronek, J. , Kralova, A. , Sekerkova, A. , Thieme, F. , & Pitha, J. (2015). Macrophage subsets in the adipose tissue could be modified by sex and the reproductive age of women. Atherosclerosis, 241, 255–258. PubMed

Kruth, H. S. , Skarlatos, S. I. , Lilly, K. , Chang, J. , & Ifrim, I. (1995). Sequestration of acetylated LDL and cholesterol crystals by human monocyte‐derived macrophages. Journal of Cell Biology, 129, 133–145. PubMed PMC

Langsted, A. , Madsen, C. M. , & Nordestgaard, B. G. (2020). Contribution of remnant cholesterol to cardiovascular risk. Journal of Internal Medicine, 288, 116–127. PubMed

Lappalainen, J. , Yeung, N. , Nguyen, S. D. , Jauhiainen, M. , Kovanen, P. T. , & Lee‐Rueckert, M. (2021). Cholesterol loading suppresses the atheroinflammatory gene polarization of human macrophages induced by colony stimulating factors. Scientific Reports, 11, 4923. PubMed PMC

Lawlor, N. , Nehar‐Belaid, D. , Grassmann, J. D. S. , Stoeckius, M. , Smibert, P. , Stitzel, M. L. , Pascual, V. , Banchereau, J. , Williams, A. , & Ucar, D. (2021). Single cell analysis of blood mononuclear cells stimulated through either LPS or anti‐CD3 and anti‐CD28. Frontiers in Immunology, 12, 636720. PubMed PMC

Liu, J. , Gillard, B. K. , Yelamanchili, D. , Gotto, A. M. , Rosales, C. , & Pownall, H. J. (2021). High free cholesterol bioavailability drives the tissue pathologies in Scarb1−/− mice. Arteriosclerosis, Thrombosis, and Vascular Biology, 41, e453‐e467. PubMed PMC

Los, D. A. , & Murata, N. (2004). Membrane fluidity and its roles in the perception of environmental signals. Biochimica et Biophysica Acta (BBA) ‐ Biomembranes, 1666, 142–157. PubMed

Nagao, T. , Qin, C. , Grosheva, I. , Maxfield, F. R. , & Pierini, L. M. (2007). Elevated cholesterol levels in the plasma membranes of macrophages inhibit migration by disrupting RhoA regulation. Arteriosclerosis, Thrombosis, and Vascular Biology, 27, 1596–1602. PubMed

Nielsen, M. C. , Andersen, M. N. , Rittig, N. , Rødgaard‐Hansen, S. , Grønbæk, H. , Moestrup, S. K. , Møller, H. J. , & Etzerodt, A. (2019). The macrophage‐related biomarkers sCD163 and sCD206 are released by different shedding mechanisms. Journal of Leukocyte Biology, 106, 1129–1138. PubMed

Ohkawa, R. , Low, H. , Mukhamedova, N. , Fu, Y. , Lai, S.‐J. , Sasaoka, M. , Hara, A. , Yamazaki, A. , Kameda, T. , Horiuchi, Y. , Meikle, P. J. , Pernes, G. , Lancaster, G. , Ditiatkovski, M. , Nestel, P. , Vaisman, B. , Sviridov, D. , Murphy, A. , Remaley, A. T. , … Tozuka, M. (2020). Cholesterol transport between red blood cells and lipoproteins contributes to cholesterol metabolism in blood. Journal of Lipid Research, 61, 1577–1588. PubMed PMC

O'Rourke, S. A. , Neto, N. G. B. , Devilly, E. , Shanley, L. C. , Fitzgerald, H. K. , Monaghan, M. G. , & Dunne, A. (2022). Cholesterol crystals drive metabolic reprogramming and M1 macrophage polarisation in primary human macrophages. Atherosclerosis, 352, 35–45. PubMed

Poledne, R. , Kralova, A. , Bartuskova, H. , Paukner, K. , Kauerova, S. , Fronek, J. , Lanska, V. , & Kralova Lesna, I. (2022). Rapid drop in coronary heart disease mortality in Czech male population—What was actually behind it? Biomedicine, 10, 2871. PubMed PMC

Poledne, R. , Kralova Lesna, I. , Kralova, A. , Fronek, J. , & Cejkova, S. (2016). The relationship between non‐HDL cholesterol and macrophage phenotypes in human adipose tissue. Journal of Lipid Research, 57, 1899–1905. PubMed PMC

Qin, C. , Nagao, T. , Grosheva, I. , Maxfield, F. R. , & Pierini, L. M. (2006). Elevated plasma membrane cholesterol content alters macrophage signaling and function. Arteriosclerosis, Thrombosis, and Vascular Biology, 26, 372–378. PubMed

Ridker, P. M. , Genest, J. , Boekholdt, S. M. , Libby, P. , Gotto, A. M. , Nordestgaard, B. G. , Mora, S. , MacFadyen, J. G. , Glynn, R. J. , & Kastelein, J. J. (2010). HDL cholesterol and residual risk of first cardiovascular events after treatment with potent statin therapy: An analysis from the JUPITER trial. Lancet, 376, 333–339. PubMed

Ross, A. C. , & Zilversmit, D. B. (1977). Chylomicron remnant cholesteryl esters as the major constituent of very low density lipoproteins in plasma of cholesterol‐fed rabbits. Journal of Lipid Research, 18, 169–181. PubMed

Smith, P. J. , Giroud, M. , Wiggins, H. L. , Gower, F. , Thorley, J. A. , Stolpe, B. , Mazzolini, J. , Dyson, R. J. , & Rappoport, J. Z. (2012). Cellular entry of nanoparticles via serum sensitive clathrin‐mediated endocytosis, and plasma membrane permeabilization. International Journal of Nanomedicine, 7, 2045–2055. PubMed PMC

Stürzebecher, P. E. , Katzmann, J. L. , & Laufs, U. (2023). What is ‘remnant cholesterol’? European Heart Journal, 44, 1446–1448. PubMed

Varbo, A. , Benn, M. , Tybjærg‐Hansen, A. , Jørgensen, A. B. , Frikke‐Schmidt, R. , & Nordestgaard, B. G. (2013). Remnant cholesterol as a causal risk factor for ischemic heart disease. Journal of the American College of Cardiology, 61, 427–436. PubMed

Wadström, B. N. , Pedersen, K. M. , Wulff, A. B. , & Nordestgaard, B. G. (2023). Elevated remnant cholesterol, plasma triglycerides, and cardiovascular and non‐cardiovascular mortality. European Heart Journal, 44, 1432–1445. PubMed

Yalcinkaya, M. , Liu, W. , Xiao, T. , Abramowicz, S. , Wang, R. , Wang, N. , Westerterp, M. , & Tall, A. R. (2024). Cholesterol trafficking to the ER leads to the activation of CaMKII/JNK/NLRP3 and promotes atherosclerosis. Journal of Lipid Research, 65, 100534. PubMed PMC