Coronavirus Research: Study Finds That SARS-CoV-2 Spike Protein Is Able To Activate Cell Signaling Pathways In Lung Vascular Cells

Coronavirus Research: Scientists from Georgetown University Medical Center-USA and the Bogomolets National Medical University-Ukraine in a new study found that vascular cells in the lung tissue begin to secrete signaling growth factors when exposed to just the spike protein of SARS-CoV-2, without the rest of the viral components. Since this signaling mechanism is potentially linked to worse outcomes in this condition, these findings could indicate new targets for the development of therapeutic agents.


 
The study team said that the mechanism of the effect of SARS-CoV-2 on human host cells has to date not been defined. The present study reports that the SARS-CoV-2 spike protein alone without the rest of the viral components is sufficient to elicit cell signaling in lung vascular cells. The treatment of human pulmonary artery smooth muscle cells or human pulmonary artery endothelial cells with recombinant SARS-CoV-2 spike protein S1 subunit (Val16 – Gln690) at 10 ng/ml (0.13 nM) caused an activation of MEK phosphorylation. The activation kinetics was transient with a peak at 10 min. The recombinant protein that contains only the ACE2 receptor binding domain of SARS-CoV-2 spike protein S1 subunit (Arg319 – Phe541), on the other hand, did not cause this activation. Consistent with the activation of cell growth signaling in lung vascular cells by SARS-CoV-2 spike protein, pulmonary vascular walls were found to be thickened in COVID-19 patients. Thus, SARS-CoV-2 spike protein-mediated cell growth signaling may participate in adverse cardiovascular/pulmonary outcomes, and this mechanism may provide new therapeutic targets to combat COVID-19.
 
The study findings were published on a preprint server and are currently being peer reviewed. https://www.biorxiv.org/content/10.1101/2020.10.12.335083v1
 
Past research have showed that patients with COVID-19 may suffer from cardiovascular and lung damage. Most individuals develop respiratory symptoms, and in a sizable minority, severe pneumonia and acute respiratory distress syndrome (ARDS) are the results. However, the elderly and those with a pre-existing disease of the cardiovascular system are peculiarly high-risk for severe and critical COVID-19.
 
This new study attempts to throw more light on how the SARS-Cov-2 virus actually affects these organs. The spike protein is considered to be key to enabling the virus to enter the host cell, engaging with the angiotensin-converting enzyme 2 (ACE2). It has two subunits, the S1 containing the ACE2 receptor-binding domain (RBD) and the S2, which mediates membrane fusion between the virus and the host cell to enable internalization of the virus following attachment.
 
The study team here shows that contrary to what is commonly believed, the spike protein alone is adequate for cell signaling in the human host. This may indicate a new mechanism whereby the virus affects the host cells. The first step was to expose human pulmonary artery smooth muscle cells to the S1 subunit for varying times. They found that at 10 ng/ml, it triggered MEK phosphorylation transiently, peaking at 10 minutes.
 
Hence this indicated the possibility that this is a cell signaling event in response to receptor binding, given the immediate response. The same response was found in endothelial cells from the human pulmonary artery. No other pathways were activated.
 
Interestingly when repeated on rat pulmonary artery smooth muscle cells, the experiment failed to produce this result, instead causing a reduction in phosphorylation which was observed as early as 10 minutes later and sustained for an hour or more.
 
The study team then pretreated the human pulmonary artery smooth muscle cells with ACE2 antibody, preventing the binding of the subsequently introduced spike protein to the ACE2 receptor. This ensured that the latter was occupied only by the antibody, but with the same result ie the activation of MEK while the viral spike protein failed to boost this signal any further. This shows that this is a specific receptor-mediated event.
 
The study team then examined the role of RBD binding. This failed to produce activation in the absence of the full-length S1 subunit. This may suggest the need to bind other spike regions to the receptor for cell signaling to be initiated.
 
Importantly the study team found that the pulmonary vessels were thickened in patients who died of COVID-19. The changes were due chiefly to tunica media thickening. The vessels tended to merge with the lung parenchyma around them, while the smooth muscle cells of the tunica media of the arteries were enlarged. Their enlarged nuclei and vacuoles are clearly observable.
 
It should be noted that patients who died of H1N1 flu do not show these changes. In fact, the wall thickness of the pulmonary arteries is ~7 μm in these patients compared to ~15 μm in COVID-19 patients – an increase of over two-fold. This results in a narrowing of the pulmonary arterial lumen in the latter.
 
The significant findings that the spike protein alone may cause deleterious cell signaling pathways to be activated in the smooth muscle and endothelium of the pulmonary vasculature indicates the effect of the infection in promoting cell growth and the replication of SARS-CoV-2.
 
Also the failure of the RBD to produce similar results indicates that possibly the cell signaling is a specific response to the binding of RBD in addition to other regions of the S1 subunit, to produce a specific cell signal transduction event. This results in inducing growth and is not just the result of nonspecific binding to the membrane receptor.
 
Although the ACE2 receptor is involved in this transduction event, the binding of the ACE2 antibody is sufficient to initiate cell signaling and not just the binding of the S1 subunit. The antibody may likely have bound to and inhibited the RBD-ACE2 binding or the cell signaling that was initiated when the antibody occupied the ACE2 receptor may have caused a reduction in the effects of the spike binding at a later time. In fact, spike-mediated signaling may well occur through a separate receptor.
 
However in rat pulmonary artery cells, the MEK/ERK pathway was not activated following the binding of the spike protein. This may explain how humans are especially severely affected by the virus. It seems that like the earlier reported ligands neuromedin and neurolysin, the SARS-CoV-2 spike protein may also cause dephosphorylation of MEK.
 
Hence the heavy toll of COVID-19 in the elderly and weak may not be just because of viral infection of host cells, but due to the activation of cell signaling pathways, as has been demonstrated here.
 
The study team hypothesizes, “SARS-CoV-2 spike protein-mediated cell signaling promotes the hyperplasia and/or hypertrophy of vascular smooth muscle and endothelial cells, contributing to the complex cardiovascular outcomes in COVID-19.”
 
Further research will be required to explain how this works and to develop better treatments.
 
The study team warned, “However a more sinister implication is that the presence of thickening of the pulmonary arteries, possibly due to hypoxia, could later lead to a higher risk of pulmonary arterial hypertension and right-sided heart failure following recovery from COVID-19. This is not seen with the earlier outbreak of SARS and may suggest that this is a feature seen only in COVID-19.”
 
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