Unraveling the pathophysiological role of angiostatin in COVID-19

ABSTRACT

Background: Angiostatin is a break-down product of plasmin(ogen). Physiologically, angiostatin is generated by platelets in an urokinase (uPA)-dependent manner. During normoxia angiostatin has anti-angiogenic/ anti-inflammatory effects and protects against lung injury, however during hypoxia/acidosis it is pro-apoptotic. In SARS-CoV infected mice, the uPA-plasminogen pathway was shown to be the most transcriptionally enriched regulating sublethal vs. lethal infection. Similarly, uPA has been shown to be transcriptionally upregulated in SARS-CoV- 2;however, the role of angiostatin has not been investigated. Aim(s) To assess role of angiostatin in COVID-19. Method(s) Plasma samples from COVID-negative controls and from hospitalized COVID-19 patients (n = 30) were collected (day 1, 7, 14, 28, 70) via the COVID-19 Surveillance Collaboration study. WHO clinical progression scale was used to assess COVID-19 severity. Angiostatin and plasminogen were quantified by immunoblot. VeroE6 cells were infected with SARS-CoV- 2 and treated with angiostatin (140 mug/ml) for 24h at pH 7.5 or 6.9. Cell death was quantified by both TUNEL and the percentage of detached cells. Immunofluorescent staining against the spike protein was used to confirm cellular infection. Result(s) Plasma angiostatin level was elevated in COVID-19 patients compared to COVID-negative controls at baseline. Both angiostatin and plasminogen increased with time of hospitalization in patients with severe COVID-19, but not with mild-to- moderate disease (p = 0.05;Fig.1). In preliminary cell culture experiments, at pH = 7.5 angiostatin decreased the percentage of detached (26 +/- 8% vs 67 +/- 5%;p = 0.0004) and TUNEL-positive VeroE6 (6 +/- 6 vs 11 +/- 7%;p = 0.07) following infection. Conversely, at pH = 6.9 angiostatin increased the percentage of detached cells following infection. Interestingly, angiostatin lowered the percentage of spike protein-positive Vero E6 at both pH (Fig.2). Conclusion(s) Angiostatin concentrations increase with disease progression in severe COVID-19. This likely reflects angiostatin's complex role in COVID-19 pathophysiology. Angiostatin promotes cell death in acidotic microenvironments (associated with severe Covid-19). Conversely, at physiological pH, angiostatin may have protective effects possibly by reducing viral entry and/or replication. (Figure Presented).