A new study by researchers from University of California – Berkeley has found that the toxic spike proteins of the SARS-CoV-2 virus are able to cause barrier dysfunction and vascular leaks in the infected, contributing to disease severity and also to various long COVID issues.
The study findings were published in the peer reviewed journal: Nature Communications.
Our study reveals the capacity and mechanism by which SARS-CoV-2 S mediates barrier dysfunction in epithelial and endothelial cells in vitro and vascular leak in vivo, thus suggesting that S alone can mediate barrier dysfunction independently from viral infection 55.
Our work indicates that levels of S observed in clinical samples from COVID-19 patients are sufficient to mediate barrier dysfunction (2.5 µg/ml) 56. Our findings suggest that, in addition to functioning in viral entry, S interactions with GAGs and integrins induce vascular leak via activation of the TGF-β pathway 18,57.
Further, our study offers a mechanistic explanation for the overproduction of TGF-β during COVID-19, which has been correlated with disease severity 58,59. Thus, our work defines a new mechanism for S-triggered barrier dysfunction and vascular leak and uncovers potential new therapeutic avenues for COVID-19 treatment.
The levels of S used in our study ranged from 2.5-20 µg/mL, with the majority of experiments conducted at 10 µg/mL (equivalent to ~50 nM). These concentrations are in line with levels detected circulating in patients as well as extrapolated from viral loads detected in sputum from critically ill COVID-19 patients (ranging from ng/mL to µg/mL levels) 56,60,61,62,63,64.
We also hypothesize that local concentrations of S accumulating in capillaries deep within tissues would likely be higher than levels circulating in patient sera. Thus, the concentrations of S we utilized in our study are consistent with circulating levels found in severe COVID-19 patients. However, the source of S that interacts with endothelial and epithelial cells to mediate barrier dysfunction during SARS-CoV-2 infection is still unclear.
Our data suggest that virion-associated full-length S, soluble trimeric S, and recombinant RBD of S are sufficient to trigger barrier dysfunction. Thus, we propose that SARS-CoV-2 can trigger barrier dysfunction through multiple avenues, including
(1) during infection of virus-permissive cells,
(2) through shedding of soluble S1 after enzymatic cleavage following ACE2 interactions on a cell,
(3) through expression of S on the surface of infected cells that can interact with neighboring cells, and
(4) through interactions with ACE2-negative non-permissive cells. Further investigation of clinical samples as well as in vivo experiments are required to explore these possibilities.
Based on our genetic data defining host factors required for S-mediated barrier dysfunction, we propose a model by which SARS-CoV-2 S first engages GAGs on the cell surface via positively charged surfaces in the RBD acquired specifically by SARS-CoV-220. Once bound to the cell surface, S can engage integrins, such as α5β1, via an RGD integrin-binding motif within the RBD18.
Engagement of integrins displaces LAP, which under steady-state conditions maintains TGF-β in an inactive state, thus resulting in the release of mature TGF-β that in turn engages the TGFBR to mediate signaling pathways regulating transient barrier dysfunction (Fig. 7K).
This compromise of barrier function is likely a result of activation of key enzymes such as HPSE, hyaluronidases, neuraminidases, MMP9, and ADAM17, which have distinct roles in disruption of the EGL and intercellular junctional complexes. Further, MMP9 and ADAM17 have separate reported roles in mediating maturation of TGF-β; thus, they may also contribute to S-mediated barrier dysfunction through this process 44,45.
This pathway is supported by the observations of others reporting a role for heparan sulfate in S cell binding, integrins in S-mediated endothelial cell activation and barrier dysfunction, and TGF-β as a correlate of COVID-19 disease severity 20,28,58,59. While this investigation begins to shed light on the mechanisms by which SARS-CoV-2 S triggers barrier disruption, further studies are required to define additional host factors as well as to determine the relative contribution of each factor to this pathway.
Our study uncovers a new potential role of S beyond ACE2 binding and viral entry, and many critical questions remain. First and foremost is how S-mediated barrier dysfunction influences outcome of SARS-CoV-2 infection.
Previous data indicate that NS1-mediated vascular leak can exacerbate a sublethal DENV infection, providing direct evidence that soluble NS1 can promote DENV pathogenesis29,65. In addition to inflammatory responses directly triggered by DENV NS165, one hypothesis by which NS1 promotes pathogenesis is through facilitating dissemination of blood-borne flaviviruses from the blood into distal tissues where the virus can replicate to high titers 32,42,55,66.
Thus, we speculate that a potential contribution of S-mediated barrier dysfunction to COVID-19 pathogenesis is to promote dissemination of SARS-CoV-2 from the lung to the blood, and then into distal organs where virus-permissive cells reside. This is exemplified by the observation that administration of S into the lungs of mice results in systemic leak in the spleen and small intestine (Fig. 3).
This could help explain the diverse clinical manifestations observed in COVID-19 patients, and in fact persistent circulation of S has been described in patients experiencing post-acute COVID-19 sequelae61. Further, the surface of the lungs is covered with a dense glycocalyx comprising many proteoglycans and glycoproteins, with a primary constituent being mucus composed of membrane-tethered and gel-forming mucins.
It has been recently demonstrated that these mucins aid cells to be refractory to SARS-CoV-2 infection due to steric hindrance of virus-cell interactions; thus, S-mediated barrier dysfunction disrupting the EGL may make virus-permissive epithelial cells more accessible to invading virus 67,68.
Our observation that S from SARS-CoV-2 but not from HCoV-229E or HCoV-OC43 triggers endothelial hyperpermeability of HPMECs suggests that the capacity to trigger barrier dysfunction in these lung cells is not conserved equivalently among all coronaviruses.
We hypothesize that the increased capacity of SARS-CoV-2 S to interact with heparan sulfate and integrins on HPMEC may explain this specificity, but additional studies are required to test this possibility 18,20. Further, the expression of ACE2 on the cell surface may influence the capacity of S proteins to interact with endothelial and epithelial cells and trigger barrier dysfunction.
This may be the case for SARS-CoV-1 and HCoV-NL63, which both utilize ACE2 as an entry receptor 69,70. The interaction of S from both SARS-CoV-1 and HCoV-NL63 with Vero-E6 cells has been shown to lead to downregulation of ACE2 expression, although via a different mechanism, which has been shown to contribute to tissue injury in the case of SARS-CoV-1 S 21,23,71.
Importantly, several reports have demonstrated that SARS-CoV-2 S can also trigger inflammatory responses and perturb barrier function in an ACE2-dependent manner 24,25,27,72,73. It will be critical to understand the relative contribution of the ACE2-independent vs. ACE2-dependent pathways to vascular leak in vivo and define which pathways a given coronavirus S protein can trigger.
Our observation that S is sufficient to mediate endothelial dysfunction and vascular leak allows a direct comparison with the flavivirus NS1 protein. Such a comparison contributes to our understanding of how viruses activate signaling pathways to mediate barrier dysfunction and leads to the concept of development of pan anti-leak therapeutics targeting multiple soluble viral proteins.
Both similarities and distinctions are apparent in the mechanisms by which S and NS1 mediate endothelial barrier dysfunction 55. Our mechanistic investigation uncovered the contributions of GAGs (HS, CS, HA), MMP9, ADAM17, HPSE, integrins, and TGF-β signaling to S-mediated barrier dysfunction.
GAG binding and activation of enzymes like MMP9, HPSE, hyaluronidase, and neuraminidases are common requirements for both NS1- and S-mediated endothelial dysfunction. In contrast, cathepsin L appears to be essential only for NS1 pathogenesis 30,31, while S-mediated dysfunction requires engagement of integrins and TGF-β signaling.
These differences may explain the distinct kinetics of our in vitro hyperpermeability assays, with NS1 causing a peak of barrier dysfunction ~6 hours post-NS1 treatment while the S-mediated peak occurs ~24 hpt with no perturbation of barrier function observed at 6 hpt.
One potential reason for this may be due to the requirement for TGF-β production and signaling as a second messenger for S-mediated endothelial dysfunction, which is not required for flavivirus NS1-induced leak. Further comparative investigations between the mechanisms of S- and NS1-mediated barrier dysfunction are needed to fully understand what makes these pathways similar yet distinct.
Although integrins and TGF-β are required for S-mediated vascular leak, their role in modulating SARS-CoV-2 infection in vivo is undoubtedly complex. For example, the well-characterized roles of integrins and TGF-β in immune cell adhesion/extravasation, modulation of inflammatory responses, and tissue repair will likely have complex effects on SARS-CoV-2 viral infection in humans 74,75.
Further, in addition to the pathogenic consequences of vascular leak in COVID-19, barrier dysfunction in the lung may also promote infiltration of immune cells that can clear virus, which would be predicted to be beneficial to the host; however, overactivation of these immune cells can lead to the “cytokine storm” typically associated with ARDS in severe COVID-19.
Dissecting the differential effects of vascular leak on SARS-CoV-2 infection in vivo will require further study. It is also important to consider that reported COVID-19 disease manifestations are diverse and may be explained by factors other than vascular leak, including pneumocyte damage resulting from immune cell infiltration and viral infection. Understanding the relative contribution of vascular leak to COVID-19 disease severity will undoubtedly be complicated but is nevertheless a critical question.
It is important to note that our working hypothesis is not that S mediates disease pathogenesis alone, but rather that the reversible vascular leak triggered by S may serve to promote viral dissemination of SARS-CoV-2 into distal tissues of infected patients, which could lead to severe disease manifestations. However, although we observe significant vascular leak in mice administered S alone, they do not overtly display signs of morbidity.
Importantly, our findings suggest that the amounts of S circulating in patients following COVID vaccination (pg/mL levels) are too low to trigger vascular leak given that our phenotype requires ng-µg/mL levels that mimic the levels observed during severe COVID-19 cases56,60.
Taken together, our study and available literature76 indicate that S-mediated vascular leak would not result from COVID-19 vaccination and therefore is not correlated with any vaccine adverse events.
In sum, our study reveals the role of S in COVID-19-associated vascular leak and provides mechanistic insight into how S mediates this process independently from viral infection and the ACE2 receptor. Although much work remains to be done to fully understand the structural basis of this mechanism as well as the implications for this pathway in SARS-CoV-2 infection and disease in humans, this work provides a foundation for future investigations by beginning to define the contribution of S to COVID-19-associated vascular leak.