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

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-1ß, 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.

Endothelial glycocalyx shields the interaction of SARS-CoV-2 spike protein with ACE2 receptors.

Endothelial cells (ECs) play a crucial role in the development and propagation of the severe COVID-19 stage as well as multiorgan dysfunction. It remains, however, controversial whether COVID-19-induced endothelial injury is caused directly by the infection of ECs with SARS-CoV-2 or via indirect mechanisms. One of the major concerns is raised by the contradictory data supporting or denying the presence of ACE2, the SARS-CoV-2 binding receptor, on the EC surface. Here, we show that primary human pulmonary artery ECs possess ACE2 capable of interaction with the viral Spike protein (S-protein) and demonstrate the crucial role of the endothelial glycocalyx in the regulation of the S-protein binding to ACE2 on ECs. Using force spectroscopy method, we directly measured ACE2- and glycocalyx-dependent adhesive forces between S-protein and ECs and characterized the nanomechanical parameters of the cells exposed to S-protein. We revealed that the intact glycocalyx strongly binds S-protein but screens its interaction with ACE2. Reduction of glycocalyx layer exposes ACE2 receptors and promotes their interaction with S-protein. These results indicate that the susceptibility of ECs to COVID-19 infection may depend on the glycocalyx condition.