KLF2 is a therapeutic target for COVID-19 induced endothelial dysfunction

  1. Suowen Xu
  2. Sihui Luo
  3. Xueying Zheng
  4. Jianping Weng

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Abstract

Coronavirus disease 2019 (COVID-19) is regarded as an endothelial disease (endothelialitis) with its mechanism being incompletely understood. Emerging evidence has demonstrated that the endothelium represents the Achilles' heel in COVID-19 patients and that endothelial dysfunction precipitates COVID-19 and accompanying multi-organ injuries. Thus, pharmacotherapies targeting endothelial dysfunction have potential to ameliorate COVID-19 and its cardiovascular complications. Primary human umbilical vein endothelial cells (HUVECs) and human pulmonary microvascular endothelial cells (HPMECs) were treated with serum from control subjects or COVID-19 patients. Downstream monocyte adhesion and associated gene/protein expression was evaluated in endothelial cells exposed to COVID-19 patient serum in the presence of KLF2 activator (Atorvastatin) or KLF2 overexpression by an adenoviral vector. Here, we demonstrate that the expression of KLF2 was significantly reduced and monocyte adhesion was increased in endothelial cells treated with COVID-19 patient serum due to elevated levels of pro-adhesive molecules, ICAM1 and VCAM1. IL-1β and TNF-α, two cytokines observed in cytokine release syndrome in COVID-19 patients, decreased KLF2 gene expression. Next-generation RNA-sequencing data showed that atorvastatin treatment leads to a cardiovascular protective transcriptome associated with improved endothelial function (vasodilation, anti-inflammation, antioxidant status, anti-thrombosis/-coagulation, anti-fibrosis and reduced angiogenesis). Treatment of HPMECs with atorvastatin or KLF2 adenovirus ameliorate COVID-19 serum-induced increase in endothelial inflammation and monocyte adhesion by increasing KLF2 expression. Altogether, the present study demonstrates that genetic and pharmacological activation of KLF2 represses COVID-19 associated endothelial dysfunction, heralding a potentially new direction to treat endothelialitis accompanying COVID-19.

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  1. ScreenIT

    SciScore for 10.1101/2021.02.20.432085: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: This study was conducted under a clinical protocol approved by the Institutional Review Board (IRB) of First Affiliated Hospital of University of Science and Technology of China (protocol number: 2020-XG(H)-009).
    Consent: All participants all agreed to participate in the study and signed informed consents approved by the IRB. 2.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableCell culture HUVECs were isolated from normal pregnant women according to our published protocols with informed consent (39).
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    HPMECs were purchased from ScienCell (Carlsbad, CA) under the identical culture conditions to HUVECs.
    HUVECs
    suggested: None
    Assay of monocyte adhesion to endothelial cells Human THP-1 monocyte adhesion assay was performed as we previously described (39, 40).
    THP-1
    suggested: CLS Cat# 300356/p804_THP-1, RRID:CVCL_0006)
    Software and Algorithms
    SentencesResources
    The sequencing data was filtered with SOAPnuke (v1.5.2) by (1) Removing reads containing sequencing adapter; (2) Removing reads whose low-quality base ratio (base quality less than or equal to 5) is more than 20%; (3) Removing reads whose unknown base ('N' base) ratio is more than 5%, afterwards clean reads were obtained and stored in FASTQ format.
    SOAPnuke
    suggested: (SOAPnuke, RRID:SCR_015025)
    The clean reads were mapped to the reference genome using HISAT2 (v2.0.4).
    HISAT2
    suggested: (HISAT2, RRID:SCR_015530)
    Bowtie2 (v2.2.5) [3] was applied to align the clean reads to the reference coding gene set， then expression level of gene was calculated by RSEM (v1.2.12).
    RSEM
    suggested: (RSEM, RRID:SCR_013027)
    The heatmap was drawn by pheatmap (v1.0.8) according to the gene expression in different samples.
    pheatmap
    suggested: (pheatmap, RRID:SCR_016418)
    Essentially, differential expression analysis was performed using the DESeq2 (v1.4.5) with Q value ≤ 0.05.
    DESeq2
    suggested: (DESeq, RRID:SCR_000154)
    To take insight to the change of phenotype, GO (http://www.geneontology.org/) and KEGG (https://www.kegg.jp/) enrichment analysis of annotated different expressed gene was performed by Phyper (https://en.wikipedia.org/wiki/Hypergeometric_distribution) based on Hypergeometric test.
    http://www.geneontology.org/
    suggested: (Mouse Genome Informatics: The Gene Ontology Project, RRID:SCR_006447)
    KEGG
    suggested: (KEGG, RRID:SCR_012773)
    Statistical analysis was performed using GraphPad Prism Software Version 8.3 (GraphPad software, La Jolla, CA).
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    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: We detected the following sentences addressing limitations in the study:
    Study limitations We recognized that the present study has two limitations. First, in light of the presence of SARS-CoV-2 viral inclusion elements within endothelial cells (3), the transcriptomic profile of SARS-CoV-2 infected human microvascular endothelial cells from different vascular beds are warranted to elucidate the gene expression profile after SARS-CoV-2 infection; Second, due to inaccessibility of post-mortem tissues from COVID-19 patients, further detection of KLF2 expression in vascular endothelium from vascular beds from small vessels (such as cardiac capillaries, arterioles and venules) and whether KLF2 expression negatively correlates with increased vascular inflammation, the severity of COVID-19, inflammatory biomarkers and coagulopathy remains to be evaluated.

    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 10.1101/2021.02.20.432085v2 on bioRxiv
  3. Version published to 10.1101/2021.02.20.432085v1 on bioRxiv