The molecular mechanisms of the plaque destabilization process in human atherosclerosis remains unsolved. Smooth Muscle Cells (SMCs) appear to be one of the most abundant cell types in atherosclerotic lesions while maintaining their high plasticity during disease progression. A better understanding of this plasticity process could reveal novel intriguing therapeutic targets to stabilize atherosclerotic lesions.
Using Single-cell RNA Sequencing (scRNA-seq) of human atherosclerotic plaques, we discovered one specific SMC subcluster we termed “Modulated SMCs.” We can show 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, PTEN) previously not connected to SMCs (or other mural cells) in atherosclerosis or vascular disease. Expression of these novel marker genes in a distinct cluster of cells has been observed by others (Bleckwehl et al., Nat. Cardiovasc. Res., 2025; Pan et al., Circulation, 2020) but lacked further description.
Knock-down (KD) of DLX6-AS1 inhibits the proliferation and migration of VSMCs 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 the inducible plaque rupture model (incomplete ligation and cuffing in ApoE-deficient mice), we can show that KD of Dlx6os1 leads to an increased plaque rupture rate and a worsened plaque phenotype.
Interestingly, the Modulated SMC subcluster seems exclusively present in carotid artery plaques and not in other vascular beds (like abdominal, thoracic and coronary artery) (Traeuble et al., Nat. Comms., 2025). We were further able to identify Modulated SMCs using spatial transcriptomics (Hybriss, Xenium) and spatial, single cell-like proteomics (deep visual proteomics) at distinct locations within advanced human lesions. Cells expressing the aforementioned specific transcripts or proteins appear abundantly located close to medial, contractile SMCs in a conversion zone towards the fibrous cap.
We can further show that plaques stemming from an inducible plaque rupture model indicate a similar expression pattern of Modulated SMC genes like Sost, Frzb, Dlx6os1, Dlx5 and Dlx6 in a distinct cell subcluster. Studies evaluating the potential role of these genes in murine atherosclerosis and plaque instability in carotid arteries are ongoing.
In summary, we show that utilizing scRNA-seq data with spatially resolved transcriptomics and proteomics approaches are powerful technologies to identify and characterize novel cell subtypes that can be linked to plasticity and fate decisions of SMCs in atherosclerosis and plaque stabilization processes. Currently these novel regulators are investigated further in vivo and in vitro.
