Single Cell Sequencing Reveals Fibroblast Heterogeneity In Healthy And Diseased Vasculature van Kuijk K1, McCracken IR2, Tillie RJHA1, Wichers Schreur R1, Taylor RS2, Dobie R3, Temmerman L1, Ramachamdran P3, Noels H4, Owens GK5, Jin H1, Wilson-Kanamori JR3, Mees BME1, Biessen EAL1, Henderson NC3, Baker AH1,3, Sluimer JC1,3 1 Cardiovascular Research Institute Maastricht, Maastricht University Medical Center, Maastricht, Netherlands, 2 BHF Centre for Cardiovascular Sciences, University of Edinburgh, Edinburgh, UK, 3 MRC centre for Inflammation Research, University of Edinburgh, Edinburgh, UK, 4 Institute for Molecular Cardiovascular Research, RWTH Aachen University, Aachen, Germany, 5 Robert M. Berne Cardiovascular Research Center, University of Virginia, Charlottesville, USA Fibroblasts are present in healthy and diseased arteries. Their role in vascular dysfunction remains unclear, as current markers are unable distinguish fibroblasts from other vascular cells. We used single cell RNA sequencing (SCS) to identify the adventitial fibroblast transcriptome and unravel heterogeneity. SCS of ~3700 CD45-/ICAM2-/PDGFRβ+ adventitial fibroblasts from healthy murine adventitia, identified a 6-gene signature, validated at protein level. PHATE dimensionality reduction predicted three differentiation trajectories, confirmed in an independent dataset. Gene ontology analysis supported divergent functional profiles: 1) vascular development, 2) antigen presentation, and 3) growth factor signaling. Immunohistochemistry confirmed protein expression of trajectory markers CD55, CXCL14 and LOX in murine and human aorta. Trajectories were driven by distinct transcription factor regulons. Transcription factor and trajectory marker expression were differentially regulated by TGFβ1, and oxidized low-density lipoprotein in fibroblasts, in vitro. Additionally, vascular dysfunction in vivo significantly increased fractions of CD55+ and CXCl14+ in PDGFRA+ cells in adventitia of 80-week vs. 10-week old murine aorta. In human atherosclerosis, trajectory 1 and 3 correlated negatively to detrimental plaque traits, e.g. plaque size and necrotic core, while trajectory 2 showed an inverse pattern. SCS provided insight in adventitial fibroblast plasticity by 3 transcriptionally divergent differentiation trajectories, regulated by transcription factors and vascular pathology. The fibroblast trajectory signatures will be instrumental to further study fibroblast function in vascular dysfunction.