SARS-CoV-2 Spike Protein-Induced Endothelial Cell Permeability

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SARS-CoV-2, the virus responsible for the COVID-19 pandemic, is a highly infectious respiratory virus that primarily targets the respiratory system. However, recent studies have suggested that the virus can also affect other systems in the body, including the cardiovascular system. Specifically, researchers have found that the spike protein of the virus, which is responsible for facilitating entry into host cells, may also contribute to endothelial cell permeability, a key factor in the development of severe COVID-19.

Endothelial cells are the cells that line the interior surface of blood vessels, and they play a critical role in regulating the exchange of molecules between the blood and tissues. Endothelial cell permeability refers to the ability of substances to pass through the endothelial cell layer, and it is regulated by a complex network of signaling pathways and protein interactions.

Recent studies have shown that the spike protein of SARS-CoV-2 can bind to the angiotensin-converting enzyme 2 (ACE2) receptor on the surface of endothelial cells, just as it does on respiratory cells. This interaction may activate a number of downstream signaling pathways, including the renin-angiotensin system (RAS), which has been implicated in the development of acute respiratory distress syndrome (ARDS) in COVID-19 patients.

In addition to activating the RAS pathway, the spike protein may also directly disrupt the integrity of the endothelial cell layer. For example, studies have shown that the spike protein can induce the release of cytokines and other inflammatory molecules that can increase endothelial cell permeability. This can lead to the leakage of fluids and proteins into surrounding tissues, which can contribute to the development of severe respiratory symptoms in COVID-19 patients.

Moreover, the spike protein-induced endothelial cell permeability may also contribute to the development of blood clots in COVID-19 patients. Endothelial cells play a critical role in maintaining the proper balance of pro- and anti-coagulant factors in the blood, and disruption of this balance can lead to the formation of blood clots. In fact, COVID-19 patients have been found to have an increased risk of blood clots, particularly in the lungs and other organs.

in a new article the researchers have found that SARS-CoV-2 spike protein S1 induces endothelial cell (EC) permeability and its vWF secretion.

reference link : https://www.mdpi.com/1422-0067/24/6/5664

It has been suggested that the SARS-CoV-2 spike protein causes not only a leaky vascular barrier but also increases the expression of vWF [22].

Thus, several clinical studies suggested that vWF is the potential biomarker of endothelial damage and thrombotic risk in COVID-19 as extremely high levels of vWF are common in COVID-19 patients [58,59].

In this study, we analyzed in vitro the SARS-CoV-2 spike protein inducing the EC permeability and vWF secretion and dissected the signaling pathway required for it. We first verified, through the assessment of the dose- or time-dependent effect, that the SARS-CoV-2 spike protein S1 RBD is sufficient to induce the EC permeability and vWF secretion.

We also assessed the proinflammatory cytokine (TNF-α, IL-6, and IL-1β) levels in RBD-treated cells as COVID-19 leads to a cytokine storm [7]. However, we only saw a negligible increase (TNF-α) or no increase in secretion (IL-6 and IL-1β) from cells treated with the RBD from 24–120 h.

We then determined the effect of various mutations (including those in SA and SC variants) in SARS-CoV-2 spike protein S1 on the EC permeability and vWF secretion. Our results suggested that the mutants we tested in this study behave like wild-type spike protein S1 in inducing the EC permeability and vWF secretion.

Since ACE2 mediates SARS-CoV-2 infection [60], we then assessed the expression of ACE2 in the RBD-treated cells and found that ACE2 expression levels were slightly elevated in the RBD-treated cells. However, a previous study [28] showed a slight reduction in ACE2 levels in pulmonary arterial ECs when treated with the spike protein.

Although the exact reason for this variation is unknown, it could be due to the difference in the spike protein treatment time and/or the cells used for the ACE2 expression analysis. A recent study identified differences, particularly in endothelial permeability, between EA.hy296 cells and primary human aortic ECs [61].

Although the use of EA.hy296 cells is a limitation of this study, most of the results we obtained for the S1 protein-induced EC permeability using EA.hy296 cells matched that obtained by using primary microvascular ECs (MVECs) by others [28,29]. Since COVID-19 is microvascular endotheliopathy, it would be interesting to confirm our findings using primary MVECs in future studies.

Since the SARS-CoV-2 nucleocapsid (N) protein circulates, like the spike protein, in COVID-19 patients and would not bind to the ACE2 receptor, it would be a good control to be included in future studies on the spike protein-induced EC permeability [30].

The excess vascular leak seen in bacterial LPS-triggered sepsis is caused by the activation of ARF6 [62]. COVID-19 causes leaky vasculature like in LPS-induced sepsis [12] and, therefore, we assessed whether ARF6 activation is altered in the RBD-treated cells. Our results suggest that the activation of ARF6, but not functionally distinct ARF1, is increased in a dose-dependent manner in the RBD-treated cells.

We used the ACE2 chemical inhibitor dalbavancin and the inhibitory antibody to analyze the role of ACE2 in the RBD-induced EC permeability and vWF secretion. Both inhibitors effectively inhibited the RBD-induced EC permeability. Chlortetracycline (the ARF6 activation inhibitor), but not QS11 (the inhibitor of ARF6 inactivation) also inhibited the EC permeability and vWF secretion, indicating that ARF6 activated in the RBD-treated cells play a role in the RBD-induced EC permeability and vWF secretion.

A previous study has shown that the SARS-CoV-2 spike protein causes endothelial dysfunction by binding to ACE2 and thereby increasing redox stress through impairing mitochondrial function [28]. In addition to this, we have shown in this study that NAC, a chemical inhibitor that reduces oxidative stress, inhibits RBD-induced EC permeability and vWF secretion.

Since NF-κB, Mtor, and dynamin play a role in SARS-CoV-2 infection and ROCK1/2 activation is important for thrombin-induced EC permeability, we also analyzed the effect of the inhibitors of these signaling molecules in the RBD-induced EC permeability and vWF-secretion. SC514 (NF-κB) and Y27632 (ROCK1/2 inhibitor) significantly inhibited the RBD-induced EC permeability and vWF secretion.

However, rapamycin and dynasore failed to reduce EC permeability and vWF secretion in the RBD-treated cells and even induced high EC permeability in the RBD-untreated cells, suggesting they may be toxic to the cells. Based on the findings of our study and that of others, we proposed a signaling cascade downstream of ACE2 for SARS-CoV-2 spike protein-induced EC permeability and its vWF secretion (Figure 8).

The SARS-CoV-2 spike protein S1 binds to ACE2 to activate ARF6 and the NF-κB signaling pathway, which may cause mitochondrial dysfunction as a result of oxidative stress promoted by ROS induction, increasing MLC-p mediated by ROCK, leading to reorganization of the actin cytoskeleton into contractile stress fibers.

This results in the opening of intercellular gaps, ultimately causing increased EC permeability and vWF secretion. Since the spike protein and cryptic SARS-CoV-2 tissue reservoirs have been suggested to be associated with the endothelial injury of long COVID [63], it is tempting to speculate that vascular permeability may mediate some of the effects of long COVID. When SARS-CoV-2 infection leads to vascular permeability, which ultimately results in the development of multi-organ tissue injury in long COVID [64].

Figure 8. Proposed schematic representation of the signaling cascade downstream of ACE2 for SARS-CoV-2 spike protein-induced EC permeability and its vWF secretion.

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