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-Kanamori; Leon J Schurgers; YH Shen; BME Mees; E A L  Biessen; N.C. Henderson; Rafael Kramann; Andrew H Baker; Judith Sluimer

doi:10.1093/cvr/cvad016

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 journal › Article › peer-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 language	English
Journal	Cardiovascular Research
DOIs	https://doi.org/10.1093/cvr/cvad016
Publication status	Published - 31 Jan 2023

Keywords / Materials (for Non-textual outputs)

Adventitia
Fibroblasts
Heterogeneity
Atherosclerosis
Single-cell RNA-Seq

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10.1093/cvr/cvad016Licence: Creative Commons: Attribution (CC-BY)

CVR-2022-0621_R2 final acceptedAccepted author manuscript, 4 MBLicence: Creative Commons: Attribution (CC-BY)
cvad016Final published version, 2.02 MBLicence: Creative Commons: Attribution (CC-BY)

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Henderson, N., Fallowfield, J. & Wigmore, S.
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van Kuijk, K., McCracken, I., Tillie, RJHA., Asselberghs, SEJ., Kheder , DA., Muitjens, S., Jin , H., Taylor, R. S., Wichers Schreur, R., Kuppe , C., Dobie, R., Ramachandran, P., Gijbels, M. J., Temmerman, L., Kirkwoord , PM., Luyten, J., Li, Y., Noels , H., Goossens , P., ... Sluimer, J. (2023). Human and murine fibroblast single cell transcriptomics reveals fibroblast clusters are differentially affected by ageing, and serum cholesterol. Cardiovascular Research. https://doi.org/10.1093/cvr/cvad016

@article{697f9c0de4bd47d3b4915fc5f7b5fd68,

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

abstract = "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 validatedplatelet-derived growth factor receptor alpha (PDGFRA) and dipeptidase 1 (DPEP1) across human and murine aorta, carotid, and femoral arteries, while traditional markers such ascluster of differentiation (CD)90 and vimentin also marked transgelin+ vascular smooth muscle cells. Next, pseudotime analysis showed multiple fibroblast clusters differentiatingalong 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 inCVD. 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 chowby 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 humansand mice. Biological relevance is evident from regulation of fibroblast clusters by age and hypercholesterolemia in vivo, associations with human atherosclerotic plaque traits, andenrichment of genes with a GWAS for CVD.",

keywords = "Adventitia, Fibroblasts, Heterogeneity, Atherosclerosis, Single-cell RNA-Seq",

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

note = "Publisher Copyright: {\textcopyright} The Author(s) 2023. Published by Oxford University Press on behalf of the European Society of Cardiology.",

year = "2023",

month = jan,

day = "31",

doi = "10.1093/cvr/cvad016",

language = "English",

journal = "Cardiovascular Research",

issn = "0008-6363",

publisher = "Elsevier",

}

van Kuijk, K, McCracken, I, Tillie, RJHA, Asselberghs, SEJ, Kheder , DA, Muitjens, S, Jin , H, Taylor, RS, Wichers Schreur, R, Kuppe , C, Dobie, R, Ramachandran, P, Gijbels, MJ, Temmerman, L, Kirkwoord , PM, Luyten, J, Li, Y, Noels , H, Goossens , P, Wilson-Kanamori, JR, Schurgers, LJ, Shen , YH, Mees, BME, Biessen, EAL, Henderson, NC, Kramann, R, Baker, AH & Sluimer, J 2023, 'Human and murine fibroblast single cell transcriptomics reveals fibroblast clusters are differentially affected by ageing, and serum cholesterol', Cardiovascular Research. https://doi.org/10.1093/cvr/cvad016

TY - JOUR

T1 - Human and murine fibroblast single cell transcriptomics reveals fibroblast clusters are differentially affected by ageing, and serum cholesterol

AU - van Kuijk, Kim

AU - McCracken, Ian

AU - Tillie, RJHA

AU - Asselberghs, SEJ

AU - Kheder , DA

AU - Muitjens, S

AU - Jin , H

AU - Taylor, R.S.

AU - Wichers Schreur, R

AU - Kuppe , C

AU - Dobie, R.

AU - Ramachandran, Prakash

AU - Gijbels, M J

AU - Temmerman, L

AU - Kirkwoord , PM

AU - Luyten, J

AU - Li, Y

AU - Noels , H

AU - Goossens , P

AU - Wilson-Kanamori, John Roger

AU - Schurgers, Leon J

AU - Shen , YH

AU - Mees, BME

AU - Biessen, E A L

AU - Henderson, N.C.

AU - Kramann, Rafael

AU - Baker, Andrew H

AU - Sluimer, Judith

PY - 2023/1/31

Y1 - 2023/1/31

N2 - 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 validatedplatelet-derived growth factor receptor alpha (PDGFRA) and dipeptidase 1 (DPEP1) across human and murine aorta, carotid, and femoral arteries, while traditional markers such ascluster of differentiation (CD)90 and vimentin also marked transgelin+ vascular smooth muscle cells. Next, pseudotime analysis showed multiple fibroblast clusters differentiatingalong 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 inCVD. 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 chowby 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 humansand mice. Biological relevance is evident from regulation of fibroblast clusters by age and hypercholesterolemia in vivo, associations with human atherosclerotic plaque traits, andenrichment of genes with a GWAS for CVD.

AB - 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 validatedplatelet-derived growth factor receptor alpha (PDGFRA) and dipeptidase 1 (DPEP1) across human and murine aorta, carotid, and femoral arteries, while traditional markers such ascluster of differentiation (CD)90 and vimentin also marked transgelin+ vascular smooth muscle cells. Next, pseudotime analysis showed multiple fibroblast clusters differentiatingalong 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 inCVD. 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 chowby 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 humansand mice. Biological relevance is evident from regulation of fibroblast clusters by age and hypercholesterolemia in vivo, associations with human atherosclerotic plaque traits, andenrichment of genes with a GWAS for CVD.

KW - Adventitia

KW - Fibroblasts

KW - Heterogeneity

KW - Atherosclerosis

KW - Single-cell RNA-Seq

U2 - 10.1093/cvr/cvad016

DO - 10.1093/cvr/cvad016

M3 - Article

SN - 0008-6363

JO - Cardiovascular Research

JF - Cardiovascular Research

ER -

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

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Keywords / Materials (for Non-textual outputs)

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