SARS-CoV-2 Spike Protein Induces Paracrine Senescence and Leukocyte Adhesion in Endothelial Cells

J Virol. 2021 Aug 10;95(17):e0079421. doi: 10.1128/JVI.00794-21. Epub 2021 Aug 10.


Increased mortality in COVID-19 cases is often associated with microvascular complications. We have recently shown that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein promotes an inflammatory cytokine interleukin 6 (IL-6)/IL-6R-induced trans signaling response and alarmin secretion. Virus-infected or spike-transfected human epithelial cells exhibited an increase in senescence, with a release of senescence-associated secretory phenotype (SASP)-related inflammatory molecules. Introduction of the bromodomain-containing protein 4 (BRD4) inhibitor AZD5153 to senescent epithelial cells reversed this effect and reduced SASP-related inflammatory molecule release in TMNK-1 or EAhy926 (representative human endothelial cell lines), when cells were exposed to cell culture medium (CM) derived from A549 cells expressing SARS-CoV-2 spike protein. Cells also exhibited a senescence phenotype with enhanced p16, p21, and senescence-associated β-galactosidase (SA-β-Gal) expression and triggered SASP pathways. Inhibition of IL-6 trans signaling by tocilizumab and inhibition of inflammatory receptor signaling by the Bruton's tyrosine kinase (BTK) inhibitor zanubrutinib, prior to exposure of CM to endothelial cells, inhibited p21 and p16 induction. We also observed an increase in reactive oxygen species (ROS) in A549 spike-transfected and endothelial cells exposed to spike-transfected CM. ROS generation in endothelial cell lines was reduced after treatment with tocilizumab and zanubrutinib. Cellular senescence was associated with an increased level of the endothelial adhesion molecules vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1), which have in vitro leukocyte attachment potential. Inhibition of senescence or SASP function prevented VCAM-1/ICAM-1 expression and leukocyte attachment. Taken together, we identified that human endothelial cells exposed to cell culture supernatant derived from SARS-CoV-2 spike protein expression displayed cellular senescence markers, leading to enhanced leukocyte adhesion. IMPORTANCE The present study was aimed at examining the underlying mechanism of extrapulmonary manifestations of SARS-CoV-2 spike protein-associated pathogenesis, with the notion that infection of the pulmonary epithelium can lead to mediators that drive endothelial dysfunction. We utilized SARS-CoV-2 spike protein expression in cultured human hepatocytes (Huh7.5) and pneumocytes (A549) to generate conditioned culture medium (CM). Endothelial cell lines (TMNK-1 or EAhy926) treated with CM exhibited an increase in cellular senescence markers by a paracrine mode and led to leukocyte adhesion. Overall, the link between these responses in endothelial cell senescence and a potential contribution to microvascular complication in productively SARS-CoV-2-infected humans is implicated. Furthermore, the use of inhibitors (BTK, IL-6, and BRD4) showed a reverse effect in the senescent cells. These results may support the selection of potential adjunct therapeutic modalities to impede SARS-CoV-2-associated pathogenesis.

Keywords: SARS-CoV-2; spike protein.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • A549 Cells
  • Cell Adhesion
  • Cell Cycle Proteins / antagonists & inhibitors
  • Cell Cycle Proteins / metabolism
  • Cellular Senescence*
  • Endothelial Cells / metabolism*
  • Endothelial Cells / pathology
  • Endothelial Cells / virology
  • Heterocyclic Compounds, 2-Ring / pharmacology
  • Humans
  • Intercellular Adhesion Molecule-1 / metabolism
  • Interleukin-6 / metabolism
  • Leukocytes / metabolism*
  • Leukocytes / pathology
  • Leukocytes / virology
  • Paracrine Communication*
  • Piperazines / pharmacology
  • Pyrazoles
  • Pyridazines
  • Reactive Oxygen Species / metabolism
  • Receptors, Interleukin-6 / metabolism
  • SARS-CoV-2 / metabolism*
  • Signal Transduction
  • Spike Glycoprotein, Coronavirus / metabolism*
  • Transcription Factors / antagonists & inhibitors
  • Transcription Factors / metabolism
  • Vascular Cell Adhesion Molecule-1 / metabolism


  • BRD4 protein, human
  • Cell Cycle Proteins
  • Heterocyclic Compounds, 2-Ring
  • ICAM1 protein, human
  • IL6 protein, human
  • IL6R protein, human
  • Interleukin-6
  • Piperazines
  • Pyrazoles
  • Pyridazines
  • Reactive Oxygen Species
  • Receptors, Interleukin-6
  • Spike Glycoprotein, Coronavirus
  • Transcription Factors
  • Vascular Cell Adhesion Molecule-1
  • spike protein, SARS-CoV-2
  • Intercellular Adhesion Molecule-1
  • AZD5153