Deregulation of vascular smooth muscle cell (VSMC) phenotypic switching is implicated in vascular disease development and VSMCs produce the majority of cells in late-stage atherosclerotic plaques. However, VSMC accumulation in mouse models of vascular disease arises from clonal expansion of very few medial cells. The source of these cells and mechanism of their selection are unknown.

Hypothesising that VSMCs which expand in disease would be distinctive within the healthy population, single cell RNA sequencing (scRNA-seq) was combined with mouse VSMC-specific lineage tracing to profile VSMC transcriptomes from healthy aorta and two in vivo disease models (CL – carotid ligation and HFD – high-fat diet-induced atherosclerosis). This identified VSMCs expressing stem cell antigen-1 (Sca1), which lacked expression of conventional VSMC markers, but were also distinct from adventitial and endothelial cell profiles. Induction of Sca1 was evident in phenotypically switched VSMCs in vitro and in both in vivo disease models. Notably, Sca1+ VSMCs were a rare subset in the healthy aorta, but more prevalent in the two disease datasets and expressed an activated, responsive gene signature in all three environments. This gene signature contains known regulators of plaque progression; suggestive that they may be candidates for those cells selectively proliferating in disease.

Focusing on VSMCs from CL, the scRNA-seq dataset lacked discrete clusters of gene expression. Instead a transition in expression profiles could be seen, allowing for cells to be mapped onto a linear trajectory, using an unbiased approach. This trajectory showed contractile marker downregulation over pseudotime, corresponding to increased expression of Sca1 and proliferation marker Ki67. Moreover, Ki67+Sca1+ VSMCs were identified post-CL via flow cytometry, indicating that Sca1+ VSMCs indeed expand in disease. Investigation of candidate genes with differential expression across this pseudotime revealed enrichment for VSMC activation and disease-relevant gene ontology pathways. Further disease relevance of these candidates was highlighted using immunostaining of mouse and human plaque sections and comparison to other scRNA-seq datasets.

Finally, a lineage traced tissue explant model was developed to study clonal VSMC proliferation in vitro. Traditional dissociated primary cell culture causes spontaneous VSMC phenotypic switching and general proliferation, which is not representative of the in vivo VSMC response. The explant model presented here maintains cell-cell and cell-extracellular matrix contact, resulting in limited proliferation of VSMCs and formation of monoclonal patches of VSMCs, comparable to those seen in vivo. Additionally, the model replicated published observations of differences in proliferation of VSMCs derived from two distinct aortic regions and increased proliferation with growth factor treatment. Lentiviral transduction of the explanted VSMCs allowed for genetic manipulation, providing a platform to test the influence of individual genes on VSMC clonal expansion.

In summary, this work identified a rare Sca1+ VSMC population which may represent a source of clonally expanding VSMCs. A comprehensive resource of transcriptional data from healthy and disease associated VSMCs has been generated and interrogation revealed commonality in activated Sca1+ VSMC expression profiles. Further investigation demonstrated a trajectory of gene expression, implicating many disease-relevant genes in VSMC activation. When combined with the in vitro lineage labelled model of VSMC proliferation, this provides a basis for identification and screening of genes involved in clonal expansion, with the ultimate aim to isolate or selectively target specific detrimental VSMC subpopulations.