The SARS-CoV-2 spike protein alters barrier function in 2D static and 3D microfluidic in vitro models of the human blood–brain barrier

  1. Tetyana P. Buzhdygan
  2. Brandon J. DeOre
  3. Abigail Baldwin-Leclair
  4. Hannah McGary
  5. Roshanak Razmpour
  6. Peter A. Galie
  7. Raghava Potula
  8. Allison M. Andrews
  9. Servio H. Ramirez

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Abstract

As researchers across the globe have focused their attention on understanding SARS-CoV-2, the picture that is emerging is that of a virus that has serious effects on the vasculature in multiple organ systems including the cerebral vasculature. Observed effects on the central nervous system includes neurological symptoms (headache, nausea, dizziness), fatal microclot formation and in rare cases encephalitis. However, our understanding of how the virus causes these mild to severe neurological symptoms and how the cerebral vasculature is impacted remains unclear. Thus, the results presented in this report explored whether deleterious outcomes from the SARS-COV-2 viral spike protein on primary human brain microvascular endothelial cells (hBMVECs) could be observed. First, using postmortem brain tissue, we show that the angiotensin converting enzyme 2 or ACE2 (a known binding target for the SARS-CoV-2 spike protein), is expressed throughout various caliber vessels in the frontal cortex. Additionally, ACE2 was also detectable in primary human brain microvascular endothelial (hBMVEC) maintained under cell culture conditions. Analysis for cell viability revealed that neither the S1, S2 or a truncated form of the S1 containing only the RBD had minimal effects on hBMVEC viability within a 48hr exposure window. However, when the viral spike proteins were introduced into model systems that recapitulate the essential features of the Blood-Brain Barrier (BBB), breach to the barrier was evident in various degrees depending on the spike protein subunit tested. Key to our findings is the demonstration that S1 promotes loss of barrier integrity in an advanced 3D microfluid model of the human BBB, a platform that most closely resembles the human physiological conditions at this CNS interface. Subsequent analysis also showed the ability for SARS-CoV-2 spike proteins to trigger a pro-inflammatory response on brain endothelial cells that may contribute to an altered state of BBB function. Together, these results are the first to show the direct impact that the SARS-CoV-2 spike protein could have on brain endothelial cells; thereby offering a plausible explanation for the neurological consequences seen in COVID-19 patients.

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  1. ScreenIT

    SciScore for 10.1101/2020.06.15.150912: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementConsent: Healthy tissue was provided (under informed consent) by the Laboratory of Developmental Biology (University of Washington, Seattle, WA) with approval granted by Temple University’s (Philadelphia, PA) Institutional Review Board and in full compliance by the National Institutes of Health’s (NIH) ethical guidelines.
    IRB: Healthy tissue was provided (under informed consent) by the Laboratory of Developmental Biology (University of Washington, Seattle, WA) with approval granted by Temple University’s (Philadelphia, PA) Institutional Review Board and in full compliance by the National Institutes of Health’s (NIH) ethical guidelines.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    The sections were subsequently incubated in rabbit anti-human ACE2 antibody (1:500, Abcam, ab15348) for 1 hr at RT.
    anti-human ACE2
    suggested: (Abcam Cat# ab15348, RRID:AB_301861)
    Positive antibody binding was detected using anti-rabbit IgG ImmPRESS-AP polymer reagent (Vector Laboratories, MP-5401) and visualized via a 10 min incubation in Vector Blue AP substrate (Vector Laboratories, SK-5300).
    anti-rabbit IgG
    suggested: (Vector Laboratories Cat# MP-5401, RRID:AB_2336536)
    MP-5401
    suggested: (Vector Laboratories Cat# MP-5401, RRID:AB_2336536)
    Cells were resuspended for 30 min in 100 μL of flow cytometry buffer and anti-ICAM-1 (Pe-Cy7, Biolegend) and anti-VCAM (APC, Biolegend) preconjugated primary antibodies.
    anti-ICAM-1
    suggested: None
    anti-VCAM (APC
    suggested: None
    Blocked protein blot was incubated with affinity-purified rabbit anti-ACE2 (1:1000, Abcam, Cat No 15348), and mouse anti-b-actin (1:5000, Sigma, Cat No A5441), in PBS with 0.05% Tween-20 and 10% Odyssey blocking buffer at 4°C overnight, followed by incubation with goat anti-rabbit IRDye 800CW IgG and goat anti-mouse IRDye 680RD secondary antibody in PBS (1:20,000) at room temperature for 1 h.
    anti-ACE2
    suggested: None
    anti-b-actin
    suggested: (Sigma-Aldrich Cat# A5441, RRID:AB_476744)
    anti-rabbit
    suggested: (LI-COR Biosciences Cat# 925-68073, RRID:AB_2716687)
    anti-mouse
    suggested: None
    Software and Algorithms
    SentencesResources
    Immunohistochemistry and Imaging: Formalin-fixed paraffin-embedded human frontal cortex brain tissue blocks of both normal and diseased origin (mixed-type dementia, cardiovascular and pulmonary dysfunction) were procured from ProteoGenex, Inc. (Inglewood, CA), and serially sectioned at a thickness of 5 µm each.
    ProteoGenex
    suggested: (ProteoGenex, RRID:SCR_013844)
    For Imaging, all sections were scanned with the Aperio AT2 slide scanner (Leica Biosystems) and analyzed using Aperio ImageScope software (v12.3.2.8013).
    ImageScope
    suggested: (ImageScope, RRID:SCR_014311)
    10,000 events per sample were acquired with a FACS BD Canto II flow cytometer (BD Biosciences) and data was then analyzed with FlowJo software (Tree Star, Ashland, OR, USA).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Band intensities were quantified using ImageJ software (NIH, Bethesda).
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    The data collected was analyzed using Prism v6.0 (GraphPad Software, San Diego, CA).
    Prism
    suggested: (PRISM, RRID:SCR_005375)
    GraphPad
    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: 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: Please consider improving the rainbow (“jet”) colormap(s) used on page 34. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    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 10.1101/2020.06.15.150912 on bioRxiv