Lack of evidence of ACE2 expression and replicative infection by SARS-CoV-2 in human endothelial cells

  1. Ian McCracken
  2. Gaye Saginc
  3. Liqun He
  4. Alik Huseynov
  5. Alison Daniels
  6. Sarah Fletcher
  7. Claire Peghaire
  8. Viktoria Kalna
  9. Maarja Andaloussi-Mäe
  10. Lars Muhl
  11. Nicky M. Craig
  12. Samantha J. Griffiths
  13. Jürgen G. Haas
  14. Christine Tait-Burkard
  15. Urban Lendahl
  16. Graeme M. Birdsey
  17. Christer Betsholtz
  18. Michela Noseda
  19. Andrew Baker
  20. Anna M. Randi

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Abstract

A striking feature of severe COVID-19 is thrombosis in large as well as small vessels of multiple organs. This has led to the assumption that SARS-CoV-2 virus directly infects and damages the vascular endothelium. However, endothelial expression of ACE2, the cellular receptor for SARS-CoV-2, has not been convincingly demonstrated. Interrogating human bulk and single-cell transcriptomic data, we found ACE2 expression in endothelial cells to be extremely low or absent in vivo and not upregulated by exposure to inflammatory agents in vitro . Also, the endothelial chromatin landscape at the ACE2 locus showed presence of repressive and absence of activation marks, suggesting that the gene is inactive in endothelial cells. Finally, we failed to achieve infection and replication of SARS-CoV-2 in cultured human endothelial cells, which were permissive to productive infection by coronavirus 229E that uses CD13 as the receptor. Our data suggest that SARS-Cov-2 is unlikely to infect endothelial cells directly; these findings are consistent with a scenario where endothelial injury is indirectly caused by the infection of neighbouring epithelial cells and/or due to systemic effects mediated by immune cells, platelets, complement activation, and/or proinflammatory cytokines.

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  1. ScreenIT

    SciScore for 10.1101/2020.12.02.391664: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    HUVEC were plated at a density of 10,000 cells/96-well plate in EGM-2 supplemented with recombinant human TNF-α (300-01A, PeproTech EC Ltd), IL1-β (200-01B, PeproTech EC Ltd), IL8 (72aa, monocytes derived, 200-08M, PeproTech EC Ltd) and IL-6/IL-6R Alpha Protein Chimera (8954-SR, R&D Biotechne) at 0, 0.01, 0.1 or 1.0 ng/ml for 4 or 24 hours.
    HUVEC
    suggested: RRID:CVCL_A4BD)
    Software and Algorithms
    SentencesResources
    Whole-transcriptome analysis with total RNA sequencing (RNA-Seq): RNA-seq data files with gene quantifications for primary human epithelial and endothelial cells were downloaded from the ENCODE database (www.encodeproject.org)6, using Bioconductor package “ENCODEExplorer” and R.
    Bioconductor
    suggested: (Bioconductor, RRID:SCR_006442)
    The data were processed using Seurat package (version: 3.1.1) in R.
    Seurat
    suggested: (SEURAT, RRID:SCR_007322)
    ChIP-seq reads were mapped to the human reference genome GRCh37/hg19.
    ChIP-seq
    suggested: (ChIP-seq, RRID:SCR_001237)
    Tracks were visualized using the UCSC Genome Browser database (https://genome.ucsc.edu).
    UCSC Genome Browser
    suggested: (UCSC Genome Browser, RRID:SCR_005780)
    HUVEC chromatin-state discovery and genome annotation was obtained using ChromHMM7 from ENCODE.
    ENCODE
    suggested: (Encode, RRID:SCR_015482)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  2. Version published to 10.1101/2020.12.02.391664 on bioRxiv