Fibroblast heterogeneity revealed by single cell sequencing in healthy and atherosclerotic arteries

Specific markers and in-depth analysis of adventitial fibroblast heterogeneity are underexplored. Using single-cell RNA-sequencing of enriched fraction of adventitial mesenchymal cells, we defined and quantified novel markers in healthy murine adventitia. PHATE dimensionality reduction and subsequent clustering characterized heterogeneity, predicting three novel differentiation trajectories. Gene ontology (GO) analysis supported divergent functional profiles, related to vascular development, antigen presentation and collagen fibril organization. Vascular pathologies, including ageing and hypercholesterolemia, differentially affected trajectory presence in vivo. We validated increased presence of the trajectory with collagen gene ontology prior to plaque development, and its association with increased adventitial collagen accumulation, while trajectory presence diminished in advanced atherosclerosis. Fibroblast trajectory gene-sets were also observed in human advanced atherosclerosis, where aforementioned trajectory negatively correlated to detrimental plaque traits. We have therefore unveiled the basis for fibroblast heterogeneity in health and vascular pathologies, and our data suggest that trajectory regulation will be instrumental for trajectory-specific interventions. Overall design: Characterisation of adventitial fibroblast heterogeneity from aorta of LDLR-ko mice on chow or high cholesterol diet using single cell RNA sequencing.