Human and murine single-cell RNA-sequencing reveal fibroblast heterogeneity in healthy and diseased vasculature and differential regulation by ageing and serum cholesterol

Background and Aims: As current markers are unable to distinguish adventitial fibroblasts from other vascular cells, we investigated their transcriptome to reveal cell type markers, heterogeneity, and regulation by cardiovascular disease (CVD) risk factors. Methods: Single-cell RNA sequencing analysis of ∼3700 CD45-/ICAM2-/PDGFRβ+ fibroblasts from healthy murine adventitia was followed by validation and regulatory studies in healthy and atherosclerotic tissue of mice and humans. Results: Immunohistochemistry and flow cytometry validated platelet-derived growth factor receptor α and dipeptidase 1 as fibroblast markers across human and murine arteries. Pseudotime analysis predicted three fibroblast differentiation trajectories, validated in an independent dataset and on protein level. Gene ontology analysis supported divergent functional profiles related to vascular development, antigen presentation or collagen fibril organization. Fibroblast trajectories were significantly enriched for genes with known genome-wide associations to CVD. CVD risk factors aging and hypercholesterolemia differentially expanded murine trajectory 2 or 3 fibroblasts respectively, coinciding with increased adventitial collagen (1.2-fold, p<0.01). Immunohistochemistry, bulk, and single-cell transcriptomics of human carotid and aorta specimens showed trajectory presence in healthy and atherosclerotic arteries, and differential correlations to atherosclerotic plaque traits. Conclusions: We provide adventitial fibroblast cell type markers and three transcriptionally divergent fibroblast differentiation trajectories. Regulation by CVD risk factors, presence in human atherosclerosis, and enrichment of genes associated with CVD, implicate their biological relevance in CVD.