A genome-wide, single-cell analysis of vascular smooth muscle cell plasticity

Vascular smooth muscle cells (VSMCs) possess a remarkable capacity to change phenotype in response to injury or inflammation. In healthy arteries, VSMCs exist in a contractile state, but upon vascular inflammation or injury, they can switch into an activated state, in which they downregulate the contractile differentiation markers and show increased migration, proliferation and secretion of proinflammatory cytokines. This process is termed phenotypic switching and can lead to VSMC accumulation within atherosclerotic plaques. Previous observations of clonal expansion of a small number of VSMCs in atherosclerosis suggested that VSMCs were functionally heterogeneous. I hypothesised that functional heterogeneity of VSMCs in disease may originate from VSMC heterogeneity in healthy arteries. In the first part of this thesis I explored the regional heterogeneity of VSMCs originating from different parts of the mouse aorta, as well as heterogeneity of VSMCs within a vascular bed using single-cell and bulk RNA sequencing. VSMCs originating from the atherosclerosis-prone aortic arch and atherosclerosis-resistant descending thoracic aorta were found to have distinct transcriptional signatures at the single-cell level. Additionally, several disease-relevant genes were observed to be heterogeneously expressed within both vascular beds. In the second chapter I identified and characterised a rare subset of VSMCs expressing Stem cell antigen 1 (SCA1). Single-cell RNA-seq was combined with VSMC-specific lineage tracing to profile gene expression in individual VSMCs from healthy mouse arteries and to compare SCA1-expressing VSMCs to other cells. SCA1-positive VSMCs were heterogeneous, with many of them expressing low levels of contractile VSMC markers. Additionally, a subset of SCA1-positive VSMCs in healthy arteries expressed transcriptional signatures characteristic of activated VSMCs involved in phenotypic switching. In the third chapter I investigated the involvement of SCA1-positive VSMCs in phenotypic switching. SCA1 upregulation was found to mark the process of VSMC phenotypic switching following in vitro culture and in vivo vascular injury. Single-cell RNA-seq profiling of VSMCs in atherosclerosis and following vascular injury showed that Ly6a/Sca1-expressing VSMCs were present and expressed transcriptional signatures similar to activated SCA1-positive cells observed in healthy arteries. Overall the results presented in this thesis highlight the heterogeneous nature of VSMCs in healthy arteries, both regionally and within a vascular bed. I identified a rare subset of SCA1-positive VSMCs with activated transcriptional signatures in healthy arteries. I hypothesised that SCA1-positive VSMCs may be responsible for clonal expansion of VSMCs in atherosclerosis, which would have clinical implications for earlier detection and specific targeting of expanding VSMCs in atherosclerosis in the future. In support of this hypothesis I have shown that Ly6a/Sca1 is upregulated in model systems of VSMC phenotypic switching and that transcriptional signatures of Ly6a/Sca1-expressing VSMCs in mouse atherosclerosis and vascular injury resemble those of healthy activated SCA1-positive VSMCs.BBSRC DTP studentshi