Tie2 activation protects against prothrombotic endothelial dysfunction in COVID-19
Alec A. Schmaier, Gabriel M. Pajares Hurtado, Zachary J. Manickas-Hill, Kelsey D. Sack, Siyu M. Chen, Victoria Bhambhani, Juweria Quadir, Anjali K. Nath, Ai-ris Y. Collier, Debby Ngo, Dan H. Barouch, Nathan I. Shapiro, Robert E. Gerszten, Xu G. Yu, MGH COVID-19 Collection and Processing Team, Kevin G. Peters, Robert Flaumenhaft, Samir M. Parikh
Alec A. Schmaier, Gabriel M. Pajares Hurtado, Zachary J. Manickas-Hill, Kelsey D. Sack, Siyu M. Chen, Victoria Bhambhani, Juweria Quadir, Anjali K. Nath, Ai-ris Y. Collier, Debby Ngo, Dan H. Barouch, Nathan I. Shapiro, Robert E. Gerszten, Xu G. Yu, MGH COVID-19 Collection and Processing Team, Kevin G. Peters, Robert Flaumenhaft, Samir M. Parikh
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Research Article COVID-19 Vascular biology

Tie2 activation protects against prothrombotic endothelial dysfunction in COVID-19

Abstract

Endothelial dysfunction accompanies the microvascular thrombosis commonly observed in severe COVID-19. Constitutively, the endothelial surface is anticoagulant, a property maintained at least in part via signaling through the Tie2 receptor. During inflammation, the Tie2 antagonist angiopoietin-2 (Angpt-2) is released from endothelial cells and inhibits Tie2, promoting a prothrombotic phenotypic shift. We sought to assess whether severe COVID-19 is associated with procoagulant endothelial dysfunction and alterations in the Tie2/angiopoietin axis. Primary HUVECs treated with plasma from patients with severe COVID-19 upregulated the expression of thromboinflammatory genes, inhibited the expression of antithrombotic genes, and promoted coagulation on the endothelial surface. Pharmacologic activation of Tie2 with the small molecule AKB-9778 reversed the prothrombotic state induced by COVID-19 plasma in primary endothelial cells. Lung autopsies from patients with COVID-19 demonstrated a prothrombotic endothelial signature. Assessment of circulating endothelial markers in a cohort of 98 patients with mild, moderate, or severe COVID-19 revealed endothelial dysfunction indicative of a prothrombotic state. Angpt-2 concentrations rose with increasing disease severity, and the highest levels were associated with worse survival. These data highlight the disruption of Tie2/angiopoietin signaling and procoagulant changes in endothelial cells in severe COVID-19. Our findings provide rationale for current trials of Tie2-activating therapy with AKB-9778 in COVID-19.

Authors

Alec A. Schmaier, Gabriel M. Pajares Hurtado, Zachary J. Manickas-Hill, Kelsey D. Sack, Siyu M. Chen, Victoria Bhambhani, Juweria Quadir, Anjali K. Nath, Ai-ris Y. Collier, Debby Ngo, Dan H. Barouch, Nathan I. Shapiro, Robert E. Gerszten, Xu G. Yu, MGH COVID-19 Collection and Processing Team, Kevin G. Peters, Robert Flaumenhaft, Samir M. Parikh

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Figure 2

Plasma from patients with COVID-19 promotes activation of coagulation on endothelial cells.

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Plasma from patients with COVID-19 promotes activation of coagulation on...
HUVECs were cultured overnight in the presence of 10% pooled plasma from patients with severe (S, ICU), moderate (Mod, non-ICU), or mild (outpatient) COVID-19 or healthy controls (HC). (A–D) Cells were analyzed for their ability to generate factor Xa or thrombin. Where indicated, cells were pretreated with Angpt-1 (300 ng/mL) or AKB-9778 (5 μM) for 30 minutes prior to incubation with plasma. (A and C) Representative experiments are depicted as mean absorbance (405 nm) or the first derivative of arbitrary fluorescence units as a function of time. (B and D) The rate of reaction for factor Xa and thrombin were converted to nM/min and U/mL, respectively, by comparison to standard curve. For factor Xa and thrombin generation assays, each data point represents the mean of 3 technical replicates, with 3–5 biologic replicates performed in total. (E and F) Cells were stained with annexin V to assess for phosphatidylserine externalization (n = 3 per group). Total fluorescent area was quantified and normalized for number of nuclei. (F) Graph represents the mean total fluorescence per 3 × 3 tile scan image ± SD. Scale bar: 50 μm. Significance was determined by 1-way ANOVA using Dunnett’s post hoc test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.