OC 22.5 - Effect of angiostatin and its neutralization on cellular infection by SARS-CoV and SARS-CoV-2

Background: Angiostatin has a complex role in COVID-19 pathophysiology. High angiostatin concentrations, such as those observed in COVID-19 patient plasma, promote cell death in acidotic microenvironments, that are characteristic of severe COVID-19. Conversely, COVID-19-relevant angiostatin concentrations reduce SARS-CoV-2 (D614G) cellular entry, most likely by interacting with abundant spike protein lysine residues. We hypothesized that 1) angiostatin inhibits cellular entry by other variants of SARS-CoV-2 as well as SARS-CoV; and 2) selective angiostatin neutralizing peptides prevent angiostatin-induced cell death, while maintaining angiostatin’s SARS-CoV-2 entry inhibiting effect.

Aims: 1) To assess if angiostatin prevents entry of other SARS-CoV-2 variants and SARS-CoV.
2) To assess effect of selective angiostatin neutralization on cell death and cellular infection by SARS-CoV-2.

Methods: HEK293-ACE2 cells were infected with replication-incompetent SARS-CoV-2 pseudo-viruses (GFP-expressing; SARS-CoV-2 Omicron, SARS-CoV Urbani) with/without angiostatin (140microg/ml) and analyzed by flow cytometry. VeroE6 cells were infected with wild-type SARS-CoV-2 and treated with angiostatin (140microg/ml) in the presence/absence of selective angiostatin neutralizing or control peptides for 24h at pH=6.9. Cell death was quantified by TUNEL and percentage of detached cells. Immunofluorescent staining against spike protein was used to confirm cellular infection.

Results: Angiostatin lowered the percentage of GFP-expressing HEK293-ACE2 (Omicron: 23±5.6 vs 1.5±0.3%, p=0.003; Urbani: 73.1±5.8 vs 7.2±4.9%, p=0.0001) (Fig.1). Angiostatin neutralizing peptides reduced % of detachment (cell death) of SARS-CoV-2-infected VeroE6 in the presence of angiostatin by approximately half compared to concentration matched peptides (Fig.2). Angiostatin reduced the percentage of TUNEL and spike protein positive cells, and these reductions were maintained in the presence of peptides.

Conclusion(s): Angiostatin inhibits cellular infection not only by multiple variants of SARS-CoV-2, but also by SARS-CoV, suggesting its importance in the pathophysiology of other SARS-like beta coronaviruses. Selective angiostatin neutralizing peptides may be potential new therapeutic strategy to reduce angiostatin-induced cell death within COVID-19 without interfering with angiostatin’s ability to reduce SARS-CoV-2 cellular infection.