Vascular smooth muscle cells (VSMCs) show pronounced heterogeneity across and within vascular beds, with direct implications for their function in injury response and atherosclerosis. Here we combine single-cell transcriptomics with lineage tracing to examine VSMC heterogeneity in healthy mouse vessels. The transcriptional profiles of single VSMCs consistently reflect their region-specific developmental history and show heterogeneous expression of vascular disease-associated genes involved in inflammation, adhesion and migration. We detect a rare population of VSMC-lineage cells that express the multipotent progenitor marker Sca1, progressively downregulate contractile VSMC genes and upregulate genes associated with VSMC response to inflammation and growth factors. We find that Sca1 upregulation is a hallmark of VSMCs undergoing phenotypic switching in vitro and in vivo, and reveal an equivalent population of Sca1-positive VSMC-lineage cells in atherosclerotic plaques. Together, our analyses identify disease-relevant transcriptional signatures in VSMC-lineage cells in healthy blood vessels, with implications for disease susceptibility, diagnosis and prevention.

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Aims Traditional markers of cell senescence including p16, Lamin B1, and senescence-associated beta galactosidase (SAβG) suggest very high frequencies of senescent cells in atherosclerosis, while their removal via ‘senolysis’ has been reported to reduce atherogenesis. However, selective killing of a variety of different cell types can exacerbate atherosclerosis. We therefore examined the specificity of senescence markers in vascular smooth muscle cells (VSMCs) and the effects of genetic or pharmacological senolysis in atherosclerosis. Methods and Results We examined traditional senescence markers in human and mouse VSMCs in vitro, and in mouse atherosclerosis. p16 and SAβG increased and Lamin B1 decreased in replicative senescence (RS) and stress-induced premature senescence (SIPS) of cultured human VSMCs. In contrast, mouse VSMCs undergoing SIPS showed only modest p16 upregulation, and proliferating mouse monocyte/macrophages also expressed p16 and SAβG. Single cell RNA-sequencing (scRNA-seq) of lineage-traced mice showed increased p16 expression in VSMC-derived cells in plaques vs. normal arteries, but p16 localized to Stem cell antigen-1 (Sca1)+ or macrophage-like populations. Activation of a p16-driven suicide gene to remove p16+ vessel wall- and/or bone marrow-derived cells increased apoptotic cells, but also induced inflammation and did not change plaque size or composition. In contrast, the senolytic ABT-263 selectively reduced senescent VSMCs in culture, and markedly reduced atherogenesis. However, ABT-263 did not reduce senescence markers in vivo, and significantly reduced monocyte and platelet counts and IL6 as a marker of systemic inflammation. Conclusions We show that genetic and pharmacological senolysis have variable effects on atherosclerosis, and may promote inflammation and non-specific effects respectively. In addition, traditional markers of cell senescence such as p16 have significant limitations to identify and remove senescent cells in atherosclerosis, suggesting that senescence studies in atherosclerosis and new senolytic drugs require more specific and lineage-restricted markers before ascribing their effects entirely to senolysis.