Fibroblast-like cells accumulate late in human coronary atherosclerosis contributing to necrotic core formation

Abstract Aims Proliferation of arterial smooth muscle cells (SMCs) and their modulation to alternative mesenchymal phenotypes is central to atherosclerotic lesion growth. It has been studied extensively in mouse models, but a detailed analysis of when and where different mesenchymal cell types accumulate in human atherosclerosis is lacking. This study mapped mesenchymal cell populations during the progression of human coronary atherosclerosis and explored their associations with disease processes in human carotid plaques. Methods and results Multiplex immunostaining protocols based on single-cell RNA sequencing-validated markers were established to detect SMCs, putatively SMC-derived mesenchymal cell subsets expressing osteoprotegerin or lumican, and macrophages in sections of left anterior descending arteries from forensic autopsies. The material comprised 44 arterial segments from 38 individuals, spanning normal intima, adaptive intimal thickening, pathological intimal thickening, and fibroatheroma. Parallel analysis of carotid endarterectomy samples allowed examination of mesenchymal cell involvement in fibrosis, calcification, and apoptosis. Validated machine learning-assisted cell classification was used to phenotype entire plaques at high microscopic resolution. The combined mesenchymal cell population constituted the majority of plaque cells at all plaque stages. Cells co-expressing contractile and mesenchymal cell markers were present in normal human coronary arteries, but mesenchymal cells lacking contractile protein expression became prominent only at the fibroatheroma stage, where fibroblast-like lumican-expressing cells localized preferentially around the necrotic core. The mesenchymal cell subtypes showed no preferential co-localization with areas of fibrosis or calcification; however, secreted osteoprotegerin was found bound to calcium deposits. Fibroblast-like, lumican-expressing cells accounted for 38–54% of all apoptotic cells for which a cell origin could be determined. Conclusion Putative SMC-derived mesenchymal cells without contractile protein expression expand at the fibroatheroma stage of coronary atherosclerosis. Fibroblast-like cells localize around the necrotic core region and account for many apoptotic cells in plaques, suggesting a role in necrotic core development.