Kyeongdae Kim1, Dahee Shim1, Jun Seong Lee2, Konstantin Zaitsev3,4, Jesse W Williams3, Ki-Wook Kim3, Man-Young Jang1, Hyung Seok Jang1, Tae Jin Yun2,5, Seung Hyun Lee1, Won Kee Yoon6, Annik Prat7, Nabil G Seidah7, Jungsoon Choi8, Seung-Pyo Lee9, Sang-Ho Yoon1, Jin Wu Nam1, Je Kyung Seong10, Goo Taeg Oh11, Gwendalyn J Randolph3, Maxim N Artyomov3, Cheolho Cheong2,5, Jae-Hoon Choi1. 1. From the Department of Life Sciences (K.K., D.S., M.-Y.J., H.S.J., S.H.L., S.-H.Y., J.W.N., J.-H.C.), College of Natural Sciences, Research Institute for Natural Sciences, Hanyang University, Seoul, Republic of Korea. 2. Laboratory of Cellular Physiology and Immunology (J.S.L., T.J.Y., C.C.), Institut de Recherches Cliniques de Montréal, Canada. 3. Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO (K.Z., J.W.W., K.-W.K., G.J.R., M.N.A.). 4. Computer Technologies Department, ITMO University, Saint Petersburg, Russia (K.Z.). 5. Division of Experimental Medicine, Department of Medicine, McGill University, Montreal, Canada (T.J.Y., C.C.). 6. Biomedical Mouse Resource Center, Korean Research Institute of Bioscience and Biotechnology (KRIBB), Chungbuk, Republic of Korea (W.K.Y.). 7. Laboratory of Biochemical Neuroendocrinology (A.P., N.G.S.), Institut de Recherches Cliniques de Montréal, Canada. 8. Department of Mathematics (J.C.), College of Natural Sciences, Research Institute for Natural Sciences, Hanyang University, Seoul, Republic of Korea. 9. Cardiovascular Center and Department of Internal Medicine, Seoul National University Hospital, Republic of Korea (S.-P.L.). 10. Laboratory of Developmental Biology and Genomics, Research Institute for Veterinary Science, College of Veterinary Medicine, Korea Mouse Phenotyping Center, Seoul National University, Republic of Korea (J.K.S.). 11. Department of Life Sciences, Immune and Vascular Cell Network Research Center, National Creative Initiatives, Ewha Womans University, Seoul, Republic of Korea (G.T.O.).
Abstract
RATIONALE: Monocyte infiltration into the subintimal space and its intracellular lipid accumulation are the most prominent features of atherosclerosis. To understand the pathophysiology of atherosclerotic disease, we need to understand the characteristics of lipid-laden foamy macrophages in the subintimal space during atherosclerosis. OBJECTIVE: We sought to examine the transcriptomic profiles of foamy and nonfoamy macrophages isolated from atherosclerotic intima. METHODS AND RESULTS: Single-cell RNA sequencing analysis of CD45+ leukocytes from murine atherosclerotic aorta revealed that there are macrophage subpopulations with distinct differentially expressed genes involved in various functional pathways. To specifically characterize the intimal foamy macrophages of plaque, we developed a lipid staining-based flow cytometric method for analyzing the lipid-laden foam cells of atherosclerotic aortas. We used the fluorescent lipid probe BODIPY493/503 and assessed side-scattered light as an indication of cellular granularity. BODIPYhiSSChi foamy macrophages were found residing in intima and expressing CD11c. Foamy macrophage accumulation determined by flow cytometry was positively correlated with the severity of atherosclerosis. Bulk RNA sequencing analysis showed that compared with nonfoamy macrophages, foamy macrophages expressed few inflammatory genes but many lipid-processing genes. Intimal nonfoamy macrophages formed the major population expressing IL (interleukin)-1β and many other inflammatory transcripts in atherosclerotic aorta. CONCLUSIONS: RNA sequencing analysis of intimal macrophages from atherosclerotic aorta revealed that lipid-loaded plaque macrophages are not likely the plaque macrophages that drive lesional inflammation.
RATIONALE: Monocyte infiltration into the subintimal space and its intracellular lipid accumulation are the most prominent features of atherosclerosis. To understand the pathophysiology of atherosclerotic disease, we need to understand the characteristics of lipid-laden foamy macrophages in the subintimal space during atherosclerosis. OBJECTIVE: We sought to examine the transcriptomic profiles of foamy and nonfoamy macrophages isolated from atherosclerotic intima. METHODS AND RESULTS: Single-cell RNA sequencing analysis of CD45+ leukocytes from murineatherosclerotic aorta revealed that there are macrophage subpopulations with distinct differentially expressed genes involved in various functional pathways. To specifically characterize the intimal foamy macrophages of plaque, we developed a lipid staining-based flow cytometric method for analyzing the lipid-laden foam cells of atherosclerotic aortas. We used the fluorescent lipid probe BODIPY493/503 and assessed side-scattered light as an indication of cellular granularity. BODIPYhiSSChi foamy macrophages were found residing in intima and expressing CD11c. Foamy macrophage accumulation determined by flow cytometry was positively correlated with the severity of atherosclerosis. Bulk RNA sequencing analysis showed that compared with nonfoamy macrophages, foamy macrophages expressed few inflammatory genes but many lipid-processing genes. Intimal nonfoamy macrophages formed the major population expressing IL (interleukin)-1β and many other inflammatory transcripts in atherosclerotic aorta. CONCLUSIONS: RNA sequencing analysis of intimal macrophages from atherosclerotic aorta revealed that lipid-loaded plaque macrophages are not likely the plaque macrophages that drive lesional inflammation.
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