SARS-CoV-2 spike protein expressing epithelial cells promotes senescence associated secretory phenotype in endothelial cells and increased inflammatory response

  1. Keith Meyer
  2. Tapas Patra
  3. Vijay Mahantesh
  4. Ranjit Ray

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Abstract

Increased mortality in COVID-19 often associates with thrombotic and microvascular complications. We have recently shown that SARS-CoV-2 spike protein promotes inflammatory cytokine IL-6/IL-6R induced trans-signaling responses which modulate MCP-1 expression in human endothelial cells. MCP-1 is secreted as a major component of the senescence associated secretory phenotype (SASP). Virus infected or Spike transfected human pulmonary epithelial cells (A549) exhibited an increase in senescence related marker proteins. TMNK; as a representative human endothelial cell line, when exposed to cell culture supernatant derived from A549 cells expressing SARS-CoV-2 spike protein (Spike CM) exhibited a senescence phenotype with enhanced p16, p21, and SA-β-galactosidase expression. Inhibition of IL-6 trans-signaling by Tocilizumab, prior to exposure of supernatant to endothelial cells, inhibited p16 and p21 induction. Likewise, inhibition of receptor signaling by Zanabrutinib or Brd4 function by AZD5153 also led to limited induction of p16 expression. Senescence lead to an enhanced level of adhesion molecule, ICAM-1 and VCAM-1 in human endothelial cells, and TPH1 attachment by in vitro assay. Inhibition of senescence or SASP function prevented ICAM/VCAM expression and leukocyte attachment. We also observed an increase in oxidative stress in A549 spike transfected and endothelial cells exposed to Spike CM. ROS generation in TMNK was reduced after treatment with the IL-6 specific inhibitor Tociliximab, and with the specific inhibitors Zanabrutinib and AZD5153. Taken together, we identified that the exposure of human endothelial cells to cell culture supernatant derived from SARS-CoV-2 spike protein expression displayed cellular senescence markers leading to enhanced leukocyte adhesion with coronary blockade potential.

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  1. ScreenIT

    SciScore for 10.1101/2021.04.16.440215: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIACUC: All live virus experiments were performed in a P3 facility approved by the Institutional Biosafety Committee.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Inhibitor treatment: Tocilizumab (Absolute Antibody), AZD5153 and Zanabrutinib (Selleckehem), ST2825 (Medchem Express)
    ST2825
    suggested: None
    After 48h, cells were fixed and γH2AX antibody (Cell Signaling) was used for IF. γH2AXwas visualized using anti-rabbit Alexa Fluor 594 (Invitrogen).
    γH2AX
    suggested: None
    anti-rabbit
    suggested: (Molecular Probes Cat# A-21207, RRID:AB_141637)
    The blot from the same run was reprobed with β-actin (Sigma) HRP conjugated antibody to compare protein load in each lane.
    β-actin
    suggested: (Sigma-Aldrich Cat# A2228, RRID:AB_476697)
    Commercially available antibodies for phospho-p38 MAPK (Thr180/Tyr182, Thr202/Tyr204),
    phospho-p38 MAPK
    suggested: None
    p-AKT (S473), p21, γ-H2AX, VCAM-1, ICAM-1 were procured from Cell Signaling Technologies, and p16 antibody was procured from Santa Cruz Biotechnology.
    p-AKT
    suggested: (Antibodies-Online Cat# ABIN461277, RRID:AB_10789159)
    S473
    suggested: None
    p21
    suggested: None
    γ-H2AX
    suggested: None
    VCAM-1
    suggested: None
    ICAM-1
    suggested: None
    p16
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cell culture and transient transfection: Transformed human lung epithelial cells (A549), liver epithelial cells (Huh7.5), liver sinusoidal endothelial cells (TMNK-1) (kindly provided by A. Soto-Gutierrez, University of Pittsburg, PA), and EAhy926 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Hyclone) containing 10% fetal bovine serum (FBS) (Sigma), 100 U of penicillin/ml, and 100 mg of streptomycin/ml (Sigma).
    Huh7.5
    suggested: RRID:CVCL_YU20)
    EAhy926
    suggested: None
    Spike transfected A549 were measured at 72 h post-transfection.
    A549
    suggested: None
    Adhesion of THP-1 cells was measured by Cytoselect Leukocyte-Endothelium Adhesion assay kit (Cell Biolabs, Inc) following suppliers protocol.
    THP-1
    suggested: CLS Cat# 300356/p804_THP-1, RRID:CVCL_0006)
    TMNK-1 cells and from SARS-CoV-2 spike expressing A549 cell culture medium in the presence or absence of inhibitors were used.
    TMNK-1
    suggested: JCRB Cat# JCRB1564, RRID:CVCL_4W79)

    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 found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    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.
    • No funding statement was detected.
    • 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 10.1101/2021.04.16.440215v1 on bioRxiv