Abstract 12343: Competing Roles of Angiostatin on SARS-CoV-2 Cellular Infection: Effects of Acidosis (original) (raw)

Circulation, 2022

Abstract

Introduction: Angiostatin - plasmin(ogen) break-down product - is generated by platelets in an urokinase(uPA)-dependent manner. Angiostatin is anti-inflammatory during normoxia, and pro-apoptotic during hypoxia/acidosis. In SARS-CoV-infected mice, the uPA-plasminogen pathway was transcriptionally enriched regulating sublethal vs. lethal infection. Similarly, uPA was transcriptionally upregulated in SARS-CoV-2; however, role of angiostatin has not been investigated. Angiostatin level progressively increases in the blood of severe COVID-19 patients and this increases risk of lethal infection. We aimed to assess role of angiostatin in COVID-19. Methods: VeroE6 cells were infected with wild-type SARS-CoV-2 and treated with angiostatin (140 μg/ml) at pH 7.5 or 6.9. Cell death was quantified by the percentage of detached cells. Immunofluorescent staining against spike protein was used to confirm cellular infection. Additionally, HEK293-ACE2 cells were infected with replication-incompetent SARS-CoV-2 Reporter Virus Particles (RVP; GFP-expressing; Wuhan-Hu-1 D614G) with/without angiostatin and analyzed by flow cytometry. Co-immunoprecipitation of angiostatin and spike protein (Alpha variant) was carried out, followed by SDS-PAGE. Results: At pH 7.5 angiostatin significantly decreased the percentage of detached (24±7 vs 58±6%, p=0.002) VeroE6 following infection, and did not have a significant effect on uninfected cells. Conversely, at pH 6.9 angiostatin alone increased the percentage of detached cells (25% detachment, p=0.014), and failed to reduce the death of infected cells (detachment:53.5±4 vs 50±5%). Angiostatin lowered the percentage of spike protein-positive VeroE6 (59±3 vs 89±3%, p<0.001) and GFP-expressing HEK293-ACE2 (7±0.5 vs 29±3, p=0.001) at both pH 7.4 and 6.9. Spike protein and angiostatin were co-immunoprecipitated. Conclusions: Angiostatin likely has a complex role in COVID-19 pathophysiology. High angiostatin concentrations such as those observed in COVID-19 promote cell death in acidotic microenvironments. Conversely, at physiological pH, angiostatin may have protective effects. Irrespective of pH, angiostatin reduced SARS-CoV-2 cellular entry, possibly by interacting with spike protein.

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