Aim: 
Deregulation of VSMC phenotypic switching is implicated in vascular disease development, producing 40-90% of atherosclerotic plaque-resident cells. However, VSMC accumulation in mouse models of atherosclerosis and vascular injury arises from clonal expansion of very few medial cells1. Here we investigated VSMC transcriptional heterogeneity, which may underlie this selective response.

Methods:
We performed single-cell RNA sequencing of dissociated aortic VSMCs from healthy mice and HFD-induced plaques, using VSMC-specific Myh11-CreERt2/Rosa-26-Confetti lineage labelling. Functional profiling utilised Sca1-GFP and Ki67-RFP reporter animals.

Results:
VSMC transcriptional profiles were clearly distinct from endothelial and adventitial cells, but displayed substantial heterogeneity, including variable levels of many disease-associated genes. We also detected a rare VSMC-lineage population expressing the progenitor marker Sca1 (VSMCSca1+) and validated their existence using Sca1-GFP animals.

Regression analysis of individual VSMCSca1+ profiles revealed activation of a ‘response’ gene signature inversely correlated with contractile VSMC markers and containing many genes known to influence plaque progression. Remarkably, this was also detected within VSMCSca1+ cells from HFD-induced atherosclerotic plaques. Moreover, we show that Sca1 upregulation is a hallmark of VSMC phenotypic switching and occurs rapidly following vascular injury in vivo, with kinetics comparable to activation of VSMC proliferation. Specific isolation of proliferating cells via Ki67-RFP showed an overlap in Sca1+ and Ki67+ VSMCs.

Conclusions:
Our data demonstrates pronounced heterogeneity in healthy VSMCs. We have identified VSMCSca1+ cells within healthy vessels lacking a conventional VSMC phenotype and expressing an activated signature also present in HFD-plaque cells. Additionally, our transcriptional data provides a basis for isolation and selective targeting of specific VSMC subpopulations. 

1. Chappell, J. et al. Circ. Res. 119, 1313–1323 (2016).

04 - SINGLE CELL PROFILING REVEALS SCA1-POSITIVE VASCULAR SMOOTH MUSCLE CELLS IN HEALTHY AND DISEASED VESSELS.