Abstract
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.
Highlights
To assess whether the S1 protein causes differences in cell viability or cytotoxicity, we incubated human pulmonary arterial endothelial cells (HPAEC) with the S1 protein at various concentrations (5–25 nM) for up to 12 h and found no changes in the cell viability of HPAEC measured by the trypan blue dye exclusion method (Figure 1A)
We analyzed S1 protein-treated cells by a cell viability assay that measures the amount of lactate dehydrogenase (LDH)
2021, 22, 9041 cells by a cell viability assay that measures the amount of lactate dehydrogenase (LDH)
Summary
COVID-19 (i.e., coronavirus disease 2019) is responsible for causing severe acute respiratory syndrome, abbreviated as SARS-CoV-2. This disease results in a wide spectrum of clinical symptoms ranging from asymptomatic infection to acute respiratory distress syndrome, multifunctional organ dysfunction, and death. The SARS-CoV-2 spike protein (S1 protein), is involved in the first critical step in the interactions between the virus and the host cell surface receptors, facilitating viral cell entry. The spike protein attaches the virus to its cellular receptor, angiotensin-converting enzyme 2 (ACE2), through a defined receptor-binding domain (RBD) on the S1 protein [1]. Anecdotal evidence further supports this mechanism of viral host cell entry since increased levels of ACE2 have a positive correlation with COVID-19 infection [2]
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