Cell-Free Hemoglobin Does Not Attenuate the Effects of SARS-CoV-2 Spike Protein S1 Subunit in Pulmonary Endothelial Cells

Int J Mol Sci. 2021 Aug 22;22(16):9041. doi: 10.3390/ijms22169041.


SARS-CoV-2 primarily infects epithelial airway cells that express the host entry receptor angiotensin-converting enzyme 2 (ACE2), which binds to the S1 spike protein on the surface of the virus. To delineate the impact of S1 spike protein interaction with the ACE2 receptor, we incubated the S1 spike protein with human pulmonary arterial endothelial cells (HPAEC). HPAEC treatment with the S1 spike protein caused disruption of endothelial barrier function, increased levels of numerous inflammatory molecules (VCAM-1, ICAM-1, IL-1β, CCL5, CXCL10), elevated mitochondrial reactive oxygen species (ROS), and a mild rise in glycolytic reserve capacity. Because low oxygen tension (hypoxia) is associated with severe cases of COVID-19, we also evaluated treatment with hemoglobin (HbA) as a potential countermeasure in hypoxic and normal oxygen environments in analyses with the S1 spike protein. We found hypoxia downregulated the expression of the ACE2 receptor and increased the critical oxygen homeostatic signaling protein, hypoxia-inducible factor (HIF-1α); however, treatment of the cells with HbA yielded no apparent change in the levels of ACE2 or HIF-1α. Use of quantitative proteomics revealed that S1 spike protein-treated cells have few differentially regulated proteins in hypoxic conditions, consistent with the finding that ACE2 serves as the host viral receptor and is reduced in hypoxia. However, in normoxic conditions, we found perturbed abundance of proteins in signaling pathways related to lysosomes, extracellular matrix receptor interaction, focal adhesion, and pyrimidine metabolism. We conclude that the spike protein alone without the rest of the viral components is sufficient to elicit cell signaling in HPAEC, and that treatment with HbA failed to reverse the vast majority of these spike protein-induced changes.

Keywords: COVID-19; bioenergetics; endothelium; proteomics; spike protein.

MeSH terms

  • Angiotensin-Converting Enzyme 2 / metabolism*
  • COVID-19 / pathology*
  • COVID-19 / virology
  • Cell Hypoxia
  • Cell Survival
  • Cells, Cultured
  • Endothelial Cells / metabolism*
  • Endothelial Cells / virology
  • Endothelium, Vascular / cytology
  • Endothelium, Vascular / pathology
  • Hemoglobins / metabolism*
  • Humans
  • Protein Subunits / metabolism
  • Pulmonary Artery / cytology
  • Pulmonary Artery / pathology
  • Reactive Oxygen Species / metabolism
  • Recombinant Proteins / metabolism
  • SARS-CoV-2 / metabolism
  • SARS-CoV-2 / pathogenicity
  • Spike Glycoprotein, Coronavirus / metabolism*


  • Hemoglobins
  • Protein Subunits
  • Reactive Oxygen Species
  • Recombinant Proteins
  • Spike Glycoprotein, Coronavirus
  • spike protein, SARS-CoV-2
  • ACE2 protein, human
  • Angiotensin-Converting Enzyme 2