Abstract Body: The process of arterial atherosclerosis is characterised by accumulation of lipids and fibrous material with accompanying inflammation. As plaques progress, they restrict blood flow and cause rupture, which results in life-threatening organ ischemia and dysfunction. Although extensively studied, a clear understanding of plaque heterogeneity and the mechanisms that trigger its destabilization remains elusive. Our study reveals the molecular microarchitecture of human carotid artery plaques, using bulk and single-cell RNA sequencing combined with single-cell spatial transcriptomics. We identified distinct plaque morphologies linked to different cell type compositions, impacting early and advanced lesion formation, as well as destabilization.
Spatial transcriptomics enabled us to localize regions of neovascularization and assign hotspots for macrophage activity within distinct cellular neighbourhoods across lesions, including a proposed gradual and locally contained transdifferentiation of HMOX1+ into TREM2+ macrophages. We further identified a specific smooth muscle cell (SMC) subcluster, which we termed “Modulated SMCs.” We can demonstrate that these specific SMCs are driven by the expression of the long non-coding RNA DLX6-AS1, as well as other genes (DLX5, DLX6, SOST, SUCNR1, THSD4, FRZB, and PTEN) that were previously not associated with SMCs (or other mural cells) in atherosclerosis or vascular disease. These cells are only present close to medial, contractile SMCs in a conversion zone towards the fibrous cap. Knock-down (KD) of DLX6-AS1 inhibits the proliferation and migration of SMCs in vitro. This effect can be rescued by the addition of PDGFbb. GO terms related to fibrosis and extracellular matrix are downregulated, whereas KD cells increase features of inflammation and foam cell formation.
In vivo, using an inducible plaque rupture model (incomplete ligation and cuffing in ApoE-deficient mice), we were able to show that KD of Dlx6os1 leads to an increased plaque rupture rate and a worsened plaque phenotype. We can further show that plaques stemming from this inducible plaque rupture model present a similar expression pattern of Modulated SMC genes like Sost, Frzb, Dlx6os1, Dlx5 and Dlx6 in a distinct cell subcluster.
Our findings provide insight into the complex heterogeneity of human atherosclerosis by unravelling location and proximity of different mural (especially SMC) and immune cell substates involved in plaque progression and vulnerability.