Elevated Angiopoietin-2 inhibits thrombomodulin-mediated anticoagulation in critically ill COVID-19 patients (preprint)

Several studies suggest that hypercoagulation and endothelial dysfunction play central roles in severe forms of COVID-19. Here, we hypothesized that the high levels of the inflammatory cytokine Angiopoietin-2 (ANGPT2) reported in hospitalized COVID-19 patients might promote hypercoagulation through ANGPT2 binding to thrombomodulin with resulting inhibition of thrombin/thrombomodulin-mediated physiological anticoagulation. We therefore investigated plasma samples taken at two timepoints from 20 critically ill COVID-19 patients in intensive care regarding ANGPT2 levels and coagulation markers in comparison with 20 healthy blood donors. We found that ANGPT2 levels were increased in the COVID-19 patients in correlation with disease severity, hypercoagulation, and mortality. To test causality, we administered ANGPT2 to wildtype mice and found that it shortened bleeding time in a tail injury model. In further support of a role for ANGPT2 in physiological coagulation, bleeding time was increased in endothelial-specific Angpt2 knockout mice. Using in vitro assays, we found that ANGPT2 inhibited thrombomodulin-mediated anticoagulation and protein C activation in human donor plasma. Our data reveal a novel mechanism for ANGPT2 in hypercoagulation and suggest that Angiopoietin-2 inhibition may be tested in the treatment of hypercoagulation in severe COVID-19, as well as in certain other conditions, including sepsis.

Lack of evidence of ACE2 expression and replicative infection by SARS-CoV-2 in human endothelial cells (preprint)

A striking feature of severe COVID-19 is thrombosis in large as well as small vessels of multiple organs. This has led to the assumption that SARS-CoV-2 virus directly infects and damages the vascular endothelium. However, endothelial expression of ACE2, the cellular receptor for SARS-CoV-2, has not been convincingly demonstrated. Interrogating human bulk and single-cell transcriptomic data, we found ACE2 expression in endothelial cells to be extremely low or absent in vivo and not upregulated by exposure to inflammatory agents in vitro. Also, the endothelial chromatin landscape at the ACE2 locus showed presence of repressive and absence of activation marks, suggesting that the gene is inactive in endothelial cells. Finally, we failed to achieve infection and replication of SARS-CoV-2 in cultured human endothelial cells, which were permissive to productive infection by coronavirus 229E that uses CD13 as the receptor. Our data suggest that SARS-Cov-2 is unlikely to infect endothelial cells directly; these findings are consistent with a scenario where endothelial injury is indirectly caused by the infection of neighbouring epithelial cells and/or due to systemic effects mediated by immune cells, platelets, complement activation, and/or proinflammatory cytokines.

LL-37 fights SARS-CoV-2: The Vitamin D-Inducible Peptide LL-37 Inhibits Binding of SARS-CoV-2 Spike Protein to its Cellular Receptor Angiotensin Converting Enzyme 2 In Vitro (preprint)

Objective: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the pathogen accountable for the coronavirus disease 2019 (COVID-19) pandemic. Viral entry via binding of the receptor binding domain (RBD) located within the S1 subunit of the SARS-CoV-2 Spike (S) protein to its target receptor angiotensin converting enzyme (ACE) 2 is a key step in cell infection. The efficient transition of the virus is linked to a unique protein called open reading frame (ORF) 8. As SARS-CoV-2 infections can develop into life threatening lower respiratory syndromes, effective therapy options are urgently needed. Several publications propose vitamin D treatment, although its mode of action against COVID-19 is not fully elucidated. It is speculated that vitamin D's beneficial effects are mediated by up regulating LL-37, a well known antimicrobial peptide with antiviral effects. Methods: Recombinantly expressed SARS-CoV-2 S protein, the extended S1 subunit (S1e), the S2 subunit (S2), the receptor binding domain (RBD), and ORF8 were used for surface plasmon resonance (SPR) studies to investigate LL-37's ability to bind to SARS-CoV-2 proteins and to localize its binding site within the S protein. Binding competition studies were conducted to confirm an inhibitory action of LL-37 on the attachment of SARS-CoV-2 S protein to its entry receptor ACE2. Results: We could show that LL-37 binds to SARS-CoV-2 S protein (LL-37/S-Strep KD = 407 nM, LL-37/S-His KD = 414 nM) with the same affinity, as SARS-CoV-2 binds to hACE2 (hACE2/S-Strep KD = 374 nM, hACE2/S-His KD = 368 nM). The binding is not restricted to the RBD of the S protein, but rather distributed along the entire length of the protein. Interaction between LL-37 and ORF8 was detected with a KD of 294 nM. Further, inhibition of the binding of S-Strep (IC50 = 735 nM), S1e (IC50 = 168 nM), and RBD (IC50 = 126 nM) to hACE2 by LL-37 was demonstrated. Conclusions: We have revealed a biochemical link between vitamin D, LL-37, and COVID-19 severity. SPR analysis demonstrated that LL-37 binds to SARS-CoV-2 S protein and inhibits binding to its receptor hACE2, and most likely viral entry into the cell. This study supports the prophylactic use of vitamin D to induce LL-37 that protects from SARS-CoV-2 infection, and the therapeutic administration of vitamin D for the treatment of COVID-19 patients. Further, our results provide evidence that the direct use of LL-37 by inhalation and systemic application may reduce the severity of COVID-19.

Pericyte-specific vascular expression of SARS-CoV-2 receptor ACE2 - implications for microvascular inflammation and hypercoagulopathy in COVID-19 patients (preprint)

Accumulating clinical observations implicate vascular inflammation as an underlying cause of coagulopathy in severely ill COVID-19 patients and it was recently suggested that SARS-CoV-2 virus particles infect endothelial cells. Here, we show that endothelial cells do not express angiotensin-converting enzyme-2 (ACE2), the SARS-CoV-2 receptor. Instead, pericytes and microvascular smooth muscle cells express ACE2 in an organotypic manner. Pericyte deficiency leads to increased endothelial expression and release of Von Willebrand factor and intravascular platelet and fibrin aggregation, suggesting that pericytes limit endothelial pro-thrombotic responses. That pericytes and not endothelial cells express ACE2 may provide important clues to the pathology of COVID-19, as pericytes are normally shielded behind an endothelial barrier and may get infected only when this barrier is compromised by COVID-19 risk factors.