Transcriptomic, Specific Marker, and Pathway Analysis of Smooth Muscle Cell Foam Cells Compared to Macrophage Foam Cells in Human Atherosclerosis
BACKGROUNDSmooth muscle cells (SMCs) are reported to contribute the majority of cholesterol-overloaded foam cells in human and mouse atheromas. However, the transcriptome, specific markers, and biologic itinerary of SMC foam cells relative to macrophage foam cells have not been determined. METHODSTranscriptomic analysis by single cell RNA sequencing (scRNA-seq) was performed on fresh coronary segments from heart transplant recipients with early to intermediate stage atherosclerosis. The gene expression pattern of a putative cluster of SMC foam cells was compared to those of cultured SMCs loaded with either aggregated low density lipoprotein (agLDL) or cholesterol bound to methyl-{beta}-cyclodextrin (Chol-M{beta}CD). Candidate markers of SMC foam cells not expressed by macrophage foam cells were validated in ours and publicly available datasets, by spatial transcriptomics and by immunofluorescence microscopy of human atheromas. Pathway analysis was performed using Gene Set Enrichment Analysis Hallmark gene sets. RESULTSSMC foam cells derived from fibromyocytes were tentatively identified using a panel of markers upregulated with in vitro cholesterol loading of SMCs. agLDL loading reproduced the same transcriptional profile, whereas Chol-M{beta}CD did not reproduce any in vivo SMC state. Top genes highly represented in SMC foam cells included SERPINE1, encoding plasminogen activator inhibitor 1 (PAI-1) and CFH, complement factor H, which were validated in further human coronary scRNA-seq datasets, by Xenium spatial transcriptomics, and by immunofluorescence microscopy. Relative to macrophage foam cells, SMC foam cells exhibit a distinct biologic itinerary, including activation of extracellular matrix, coagulation and angiogenesis pathways. CONCLUSIONSSMC foam cells, which are derived from fibromyocytes ("lipomyocytes"), exhibit unique markers and biologic programs that differ markedly from macrophage foam cells in atherosclerotic plaque development. Further understanding of the role of lipomyocytes and their expression of CFH and PAI-1 expression in plaque biology may offer novel therapeutic options to reduce ischemic cardiovascular disease. Novelty and SignificanceO_ST_ABSWhat is Known?C_ST_ABSO_LISingle-cell transcriptomic studies have demonstrated vascular SMCs undergo extensive phenotypic modulation during atherosclerosis, giving rise to fibromyocytes and other intermediate cell states. C_LIO_LISMC-derived foam cells constitute a major proportion of plaque foam cells. C_LIO_LIPrevious studies have shown that SMC-derived foam cells differ from macrophage-derived foam cells in cholesterol handling and lipid metabolism, making their identification using conventional macrophage-associated foam-cell markers challenging, and highlighting the need for SMC foam cell-specific molecular markers. C_LI What New Information Does This Article ContributeO_LIBy integrating published transcriptomic datasets with bulk RNA-sequencing of agLDL-loaded human SMCs, we identify the transcriptional program of human SMC-derived foam cells and demonstrate that these cells are embedded within the fibromyocyte population rather than forming a distinct cell cluster. C_LIO_LIagLDL loading in vitro reproduces the in vivo SMC foam-cell phenotype, whereas loading with cholesterol bound to cyclodextrin induces an inflammatory state not found in any in vivo SMC state in human atherosclerosis. C_LIO_LICFH and SERPINE1 were identified as specific markers of SMC-derived foam cells and validated by immunostaining and Xenium spatial transcriptomics, providing robust markers for identification of these cells in human atherosclerotic plaques. C_LIO_LIThe top transcriptional programs activated in SMC foam cells differ completely from macrophage foam cells and include extracellular matrix remodeling, complement regulation, coagulation and angiogenesis. C_LI In this study, we mapped smooth muscle foam cells to the fibromyocyte cluster of arterial SMC subtypes in human coronary artery atheromas. In vitro loading of smooth muscle cells with aggregated low-density lipoprotein, the presumed physiologic substrate for foam cell formation during plaque development, induced a gene expression pattern that localized to the same fibromyocyte cluster. The proposed sequence of SMC foam cell formation is that fibromyocytes present in human pre-atherosclerotic intima generate the proteoglycans that bind lipoproteins and allow their uptake by fibromyocytes to generate cholesterol-loaded "lipomyocytes". Lipid loading of fibromyocytes drives expression of specific markers and transcriptional programs, but with an overall gene expression pattern that continues to resemble fibromyocytes. We demonstrate here that lipomyocytes express the specific markers CFH and SERPINE1, and exhibit activation of a completely different set of pathways relative to macrophage foam cells in the plaque. These findings provide evidence for the unique phenotype and specific markers of lipomyocytes relative to macrophage foam cells in human atherosclerosis, which will be critical in evaluating the response of these different types of foam cell to novel therapies.