Human and murine fibroblast single cell transcriptomics reveals fibroblast clusters are differentially affected by ageing, and serum cholesterol

Kim van Kuijk, Ian McCracken, RJHA Tillie, SEJ Asselberghs, DA Kheder , S Muitjens, H Jin , R.S. Taylor, R Wichers Schreur, C Kuppe , R. Dobie, Prakash Ramachandran, M J Gijbels, L Temmerman, PM Kirkwoord , J Luyten, Y Li, H Noels , P Goossens , John Roger Wilson-KanamoriLeon J Schurgers, YH Shen , BME Mees, E A L Biessen, N.C. Henderson, Rafael Kramann, Andrew H Baker, Judith Sluimer*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract / Description of output

Aims: Specific fibroblast markers and in-depth heterogeneity analysis are currently lacking, hindering functional studies in cardiovascular diseases (CVD). Here, we established cell-type markers and heterogeneity in murine and human arteries and studied the adventitial fibroblast response to CVD and its risk factors hypercholesterolemia and aging.

Methods & Results: Murine aorta scRNA-seq analysis of adventitial mesenchymal cells identified fibroblast-specific markers. Immunohistochemistry and flow cytometry validated
platelet-derived growth factor receptor alpha (PDGFRA) and dipeptidase 1 (DPEP1) across human and murine aorta, carotid, and femoral arteries, while traditional markers such as
cluster of differentiation (CD)90 and vimentin also marked transgelin+ vascular smooth muscle cells. Next, pseudotime analysis showed multiple fibroblast clusters differentiating
along trajectories. Three trajectories, marked by CD55 (Cd55+), Cxcl chemokine (Cxcl14+) and lysyl oxidase (Lox+), were reproduced in an independent RNAseq dataset.
Gene ontology analysis showed divergent functional profiles of the three trajectories, related to vascular development, antigen presentation and/or collagen fibril organization,
respectively. Trajectory-specific genes included significantly more genes with known genome-wide associations (GWAS) to CVD than expected by chance, implying a role in
CVD. Indeed, differential regulation of fibroblast clusters by CVD risk factors was shown in adventitia of aged C57BL/6J mice, and mildly hypercholesterolemic LDLR-KO mice on chow
by flow cytometry. The expansion of collagen-related CXCL14+ and LOX+ fibroblasts in aged and hypercholesterolemic aortic adventitia respectively, coincided with increased adventitial collagen. Immunohistochemistry, bulk, and single-cell transcriptomics of human carotid and aorta specimens emphasized translational value as CD55+, CXCL14+ and LOX+ fibroblasts were observed in healthy and atherosclerotic specimens. Also, trajectory-specific gene sets differentially correlated to human atherosclerotic plaque traits.

Conclusion: We provide two adventitial fibroblast-specific markers, PDGFRA and DPEP1, and demonstrate fibroblast heterogeneity in health and cardiovascular disease in humans
and mice. Biological relevance is evident from regulation of fibroblast clusters by age and hypercholesterolemia in vivo, associations with human atherosclerotic plaque traits, and
enrichment of genes with a GWAS for CVD.

Original languageEnglish
JournalCardiovascular Research
Publication statusPublished - 31 Jan 2023

Keywords / Materials (for Non-textual outputs)

  • Adventitia
  • Fibroblasts
  • Heterogeneity
  • Atherosclerosis
  • Single-cell RNA-Seq


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