Single-cell multimodal profiling of atherosclerosis identifies CD200 as a cell surface lineage marker of vascular smooth muscle cells and their derived cells

Vascular smooth muscle cells (VSMCs) play a central role in the development of atherosclerosis due in part to their capability to phenotypically transition into either a protective or harmful state. However, the ability to identify and trace VSMCs and their progeny in vivo is limited due to the lack of well-defined VSMC cell surface markers. Therefore, investigations into VSMC fate must utilize lineage-tracing mouse models, which are time-consuming and challenging to generate and not feasible in humans. Here, we employed CITE-seq to characterize the phenotypic expression of 119 cell surface proteins in mouse atherosclerosis. We found that CD200 is a highly expressed and specific marker of VSMCs, which persists even with phenotypic modulation. We validated our findings using a combination of flow cytometry, qPCR, and immunohistochemistry, all confirming that CD200 can identify and mark VSMCs and their derived cells in early to advanced mouse atherosclerotic lesions. Additionally, we describe a similar expression pattern of CD200 in human coronary and carotid atherosclerosis. Thus, our data support the use of CD200 as a lineage marker for VSMCs and VSMC-derived cells in mouse and human atherosclerosis. Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) was used to perform in-depth cell surface phenotyping of all cells within mouse atherosclerotic lesions. Aortas from ROSA26LSL-ZsGreen1/+Myh11-CreERT2 vascular smooth muscle cell lineage traced mice at various times of western diet feeding (0, 8, 16, and 26 weeks) were digested in an enzyme cocktail containing 4U/mL Liberase, 60U/mL Hyaluronidase and 60U/mL DNase in RPMI for 1 hour. Cell suspension was then labeled with the TotalSeq-A Mouse Universal Cocktail (BioLegend: 199901) oligo-conjugated antibodies and subsequently fluorescently activated cell sorted and viable cells were identified as being DRAQ5+ and DAPI-. Cells were then submitted for single cell profiling using the 10x Genomics 3’ v3 protocol. Single cell analysis identified CD200 as a potential cell surface lineage marker for VSMCs and their derived cells. To validate this, we used flow cytometry to isolate CD200+ and CD200- cells and performed qPCR on isolated cells.