Abstract Body: The fibrous cap of atherosclerotic plaques is essential for plaque stability. Rupture of the fibrous cap leads to heart attacks and strokes, and causes tens of millions of deaths globally every year. Identifying and understanding the cellular origins and plasticity of the fibrous cap is critical to developing therapeutic strategies to stabilize the atherosclerotic plaques. The fibrous cap is thought to arise oligoclonal from medial smooth muscle cells (SMCs), but whether all SMCs can give rise to the fibrous cap is unknown. Furthermore, conflicting data exist regarding whether plaque cells deeper in the lesion can give rise to the fibrous cap or vice vera.
Murine SMC-lineage traced scRNAseq data revealed a transcriptomically distinct population of Notch3 and Elastin high population of cells that localizes to the fibrous cap. Utilizing a lineage tracing mouse model driven by endogenous Notch3, we demonstrated that fibrous cap cells arise from a predefined population of SMC that expresses Notch3 at baseline. After pulse-labeling the Notch3^CreERT2; ROSA^lsl-tdTomato; Apoe^-/- mice with tamoxifen before high fat diet and then feeding them with high fat diet for 16 weeks, Notch3-lineage traced cells stain positive for SMC markers (Tagln, Cnn1) and are nearly exclusively found at the fibrous cap in multiple atherosclerotic prone beds. Furthermore, Notch3-lineage traced SMCs and chondrogenic SMCs are mutually exclusive in the plaque, as demonstrated by the minimal overlap of tdTomato with chondrogenic SMC markers, including Col2a1 and Sox9. The Notch3 lineage labeled fibrous cap-SMCs display different inflammatory and extracellular matrix program from the non-labeled SMC progenies, as demonstrated by single cell transcriptomic sequencing. Consistently, Notch3-lineage traced cells are committed to the fibrous cap fate and excluded from the calcified portions of the lesion and acellular core.
Altogether, these lineage tracing studies highlight previously unrecognized medial SMC heterogeneity in healthy vessels. Unique Notch3⁺ populations of SMCs in normal media are fated to form the lesion cap. Once the Notch3 program is turned on, cells are locked into a fibrous cap fate and do not give rise to osteochondrogenic SMCs. Importantly, Notch3^CreERT2 mice can be used as a cap-specific genetic manipulation tool to further elucidate the role of fibrous cap specific genetic programs.