Resistance of endothelial cells to SARS-CoV-2 infection in vitro

  1. Blerina Ahmetaj-Shala
  2. Thomas P. Peacock
  3. Laury Baillon
  4. Olivia C. Swann
  5. Hime Gashaw
  6. Wendy S. Barclay
  7. Jane A. Mitchell

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Abstract

Rationale

The secondary thrombotic/vascular clinical syndrome of COVID-19 suggests that SARS-CoV-2 infects not only respiratory epithelium but also the endothelium activating thrombotic pathways, disrupting barrier function and allowing access of the virus to other organs of the body. However, a direct test of susceptibility to SARS-CoV-2 of authentic endothelial cell lines has not been performed.

Objective

To determine infectibility of primary endothelial cell lines with live SARS-CoV-2 and pseudoviruses expressing SARS-CoV-2 spike protein.

Methods and Results

Expression of ACE2 and BSG pathways genes was determined in three types of endothelial cells; blood outgrowth, lung microvascular and aortic endothelial cells. For comparison nasal epithelial cells, Vero E6 cells (primate kidney fibroblast cell line) and HEK 293T cells (human embryonic kidney cells) transfected with either ACE2 or BSG were used as controls. Endothelial and Vero E6 cells were treated with live SARS-CoV-2 virus for 1 hour and imaged at 24 and 72 hours post infection. Pseudoviruses containing SARS-CoV-2, Ebola and Vesicular Stomatis Virus glycoproteins were generated and added to endothelial cells and HEK 239Ts for 2 hours and infection measured using luminescence at 48 hours post infection. Compared to nasal epithelial cells, endothelial cells expressed low or undetectable levels of ACE2 and TMPRSS2 but comparable levels of BSG, PPIA and PPIB. Endothelial cells showed no susceptibility to live SARS-CoV-2 or SARS-CoV-2 pseudovirus (but showed susceptibility to Ebola and Vesicular Stomatitis Virus). Overexpression of ACE2 but not BSG in HEK 239T cells conferred SARS-CoV-2 pseudovirus entry. Endothelial cells primed with IL-1ß remained resistant to SARS-CoV-2.

Conclusion

Endothelial cells are resistant to infection with SARS-CoV-2 virus, in line with relatively low levels of ACE2 and TMPRSS2, suggesting that the vascular dysfunction and thrombosis seen in severe COVID-19 is a result of factors released by adjacent infected cells (e.g. epithelial cells) and/or circulating, systemic inflammatory mediators.

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  1. ScreenIT

    SciScore for 10.1101/2020.11.08.372581: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    BlindingFor quantitative analysis, all images were blinded and independently scored between 1-5, where 1= 0-2, 2= 3-5, 3=6-8, 4= 9-10 and 5= >10 nucleocapsid stained cells.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Following 3x PBS washes, cells were incubated with secondary antibodies (anti-rabbit 488, anti-mouse 594) diluted 1:500 and DAPI (diluted 1 in 1000) in PBS with 2% BSA at room temperature for 1 hour in the dark.
    anti-rabbit
    suggested: None
    anti-mouse 594
    suggested: (Novus Cat# NB500-594, RRID:AB_10002685)
    Membranes were probed with the primary antibodies: mouse anti-FLAG (Sigma: F1804); mouse anti-tubulin (abcam; ab7291); and rabbit anti-CD147 (abcam; ab108308)
    anti-FLAG
    suggested: (Sigma-Aldrich Cat# F1804, RRID:AB_262044)
    anti-tubulin
    suggested: (Abcam Cat# ab7291, RRID:AB_2241126)
    anti-CD147
    suggested: (Abcam Cat# ab108308, RRID:AB_10861230)
    Near infra-red (NIR) secondary antibodies, IRDye® 680RD Goat anti-mouse (abcam; ab216776) and IRDye® 800CW Goat anti-rabbit (abcam; ab216773)) were subsequently used to detect primary antibodies.
    anti-mouse
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    African green monkey (Vero E6) cells (ATCC) and human embryonic kidney cells (293T) - 293T(ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% foetal bovine serum (FBS), 1% non-essential amino acids (NEAA) and 1% penicillin-streptomycin (P/S; Gibco).
    Vero E6
    suggested: RRID:CVCL_XD71)
    HEK 293T-ACE2 cells were produced as previous described17 (Figure 3, Supplementary Figure 2) and maintained in HEK 293T media supplemented with 1 μg/ml of puromycin.
    HEK 293T-ACE2
    suggested: None
    HEK 293T
    suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
    For transient transfections HEK 293T cells seeded in 10cm2 plates were transfected with 1 μg of empty pCAGGs vector, pCAGGs-ACE2-FLAG, or pCAGGS-BSG (synthesised by GeneArt; ThermoFisher), 24 hours later cells were transferred to 96 well plates or lysed for western blot analysis.
    293T
    suggested: None
    Software and Algorithms
    SentencesResources
    For transient transfections HEK 293T cells seeded in 10cm2 plates were transfected with 1 μg of empty pCAGGs vector, pCAGGs-ACE2-FLAG, or pCAGGS-BSG (synthesised by GeneArt; ThermoFisher), 24 hours later cells were transferred to 96 well plates or lysed for western blot analysis.
    GeneArt
    suggested: None
    ThermoFisher
    suggested: (ThermoFisher; SL 8; Centrifuge, RRID:SCR_020809)
    All images were analysed and prepared using FIJI software19.
    FIJI
    suggested: (Fiji, RRID:SCR_002285)
    Data analysis: All data were analysed on GraphPad Prism v8 and are shown as individual data points and/or mean +/− standard error of the mean (SEM) for samples as described in the figure legends.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)

    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: We detected the following sentences addressing limitations in the study:
    While we have worked to the highest standards with empirical virology protocols, there are limitations in our approach, and we cannot definitively conclude that endothelial cells are non-susceptible to infection by SARS-CoV-2 in some individuals or in some highly specific conditions in vivo. This is because the assay systems we used did not take account of critical factors present in at risk populations and/or at the site of inflammation. In an attempt to take account of basic inflammatory conditions we performed experiments in cells primed with IL-1ß, which did not confer infectability to any of our endothelial lines. However, as we find more about the complex mix of inflammatory mediators present in the lung and circulation in COVID-19 and the specific biological factors that predispose certain groups of individuals to severe disease, these can be recapitulated within in vitro assay systems. Nonetheless what our study does prove is that if endothelial cells are susceptible to SARS-CoV-2 at some distant point in the natural history of COVID-19, the pathways of viral entry are more complex than for airway epithelium.

    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 10.1101/2020.11.08.372581v1 on bioRxiv