Diverse cellular architecture of atherosclerotic plaque derives from clonal expansion of a few medial SMCs

Fibrous cap smooth muscle cells (SMCs) protect atherosclerotic lesions from rupturing and causing thrombosis, while other plaque SMCs may have detrimental roles in plaque development. To gain insight into recruitment of different plaque SMCs, we mapped their clonal architecture in aggregation chimeras of eGFP+Apoe-/- and Apoe-/- mouse embryos and in mice with a mosaic expression of fluorescent proteins in medial SMCs that were rendered atherosclerotic by PCSK9-induced hypercholesterolemia. Fibrous caps in aggregation chimeras were found constructed from large, endothelial-aligned layers of either eGFP+ or nonfluorescent SMCs, indicating substantial clonal expansion of a few cells. Similarly, plaques in mice with SMC-restricted Confetti expression showed oligoclonal SMC populations with little intermixing between the progeny of different medial SMCs. Phenotypes comprised both ACTA2+ SMCs in the cap and heterogeneous ACTA2- SMCs in the plaque interior, including chondrocyte-like cells and cells with intracellular lipid and crystalline material. Fibrous cap SMCs were invariably arranged in endothelium-aligned clonal sheets, confirming results in the aggregation chimeras. Analysis of the clonal structure showed that a low number of local medial SMCs partake in atherosclerosis and that single medial SMCs can produce several different SMC phenotypes in plaque. The combined results show that few medial SMCs proliferate to form the entire phenotypically heterogeneous plaque SMC population in murine atherosclerosis.The study was funded by grants from the Danish Independent Research Council (Sapere Aude Programme, 4004-00459B) and the Ministerio de Economia, Industria e Competividad (MEIC) with cofunding from the Fondo Europeo de Desarrollo Regional (FEDER) (SAF2016-75580-R) and by scholarships from the Danish Independent Research Council (to MBL) and the Novo Scholarship Program (to KJ). The CNIC is supported by MEIC and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505). We would like to thank Leticia Gonzalez, Lisa Maria Roge, and Dorte Wilhart Qualmann for histology and genotyping; Lisbeth Ahm Hansen and Peter Kragh for embryo work; the CNIC microscopy Unit (Veronica Labrador, Elvira Arza, and Antonio Santos-Beneit) for support on confocal microscopy; and the CNIC Histopathology Unit (Roisin Doohan, Brenda Guijarro, and Antonio Molina) for histological stainings and tissue scans.S