Cathepsin K cleavage of angiopoietin-2 creates detrimental Tie2 antagonist fragments in sepsis
Takashi Suzuki, Erik Loyde, Sara Chen, Valerie Etzrodt, Temitayo O. Idowu, Amanda J. Clark, Marie Christelle Saade, Brenda Mendoza Flores, Shulin Lu, Gabriel Birrane, Vamsidhara Vemireddy, Benjamin Seeliger, Sascha David, Samir M. Parikh
Takashi Suzuki, Erik Loyde, Sara Chen, Valerie Etzrodt, Temitayo O. Idowu, Amanda J. Clark, Marie Christelle Saade, Brenda Mendoza Flores, Shulin Lu, Gabriel Birrane, Vamsidhara Vemireddy, Benjamin Seeliger, Sascha David, Samir M. Parikh
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Research Article Inflammation Vascular biology

Cathepsin K cleavage of angiopoietin-2 creates detrimental Tie2 antagonist fragments in sepsis

Abstract

Elevated angiopoietin-2 is associated with diverse inflammatory conditions, including sepsis, a leading global cause of mortality. During inflammation, angiopoietin-2 antagonizes the endothelium-enriched receptor Tie2 to destabilize the vasculature. In other contexts, angiopoietin-2 stimulates Tie2. The basis for context-dependent antagonism remains incompletely understood. Here, we show that inflammation-induced proteolytic cleavage of angiopoietin-2 converts this ligand from Tie2 agonist to antagonist. Conditioned media from stimulated macrophages induced endothelial angiopoietin-2 secretion. Unexpectedly, this was associated with reduction of the 75 kDa full-length protein and appearance of new 25 and 50 kDa C-terminal fragments. Peptide sequencing proposed cathepsin K as a candidate protease. Cathepsin K was necessary and sufficient to cleave angiopoietin-2. Recombinant 25 and 50 kDa angiopoietin-2 fragments (cANGPT225 and cANGPT250) bound and antagonized Tie2. Cathepsin K inhibition with the phase 3 small-molecule inhibitor odanacatib improved survival in distinct murine sepsis models. Full-length angiopoietin-2 enhanced survival in endotoxemic mice administered odanacatib and, conversely, increased mortality in the drug’s absence. Odanacatib’s benefit was reversed by heterologous cANGPT225. Septic humans accumulated circulating angiopoietin-2 fragments, which were associated with adverse outcomes. These results identify cathepsin K as a candidate marker of sepsis and a proteolytic mechanism for the conversion of angiopoietin-2 from Tie2 agonist to antagonist, with therapeutic implications for inflammatory conditions associated with angiopoietin-2 induction.

Authors

Takashi Suzuki, Erik Loyde, Sara Chen, Valerie Etzrodt, Temitayo O. Idowu, Amanda J. Clark, Marie Christelle Saade, Brenda Mendoza Flores, Shulin Lu, Gabriel Birrane, Vamsidhara Vemireddy, Benjamin Seeliger, Sascha David, Samir M. Parikh

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Figure 3

Cathepsin K is responsible for cleaving ANGPT2 into Tie2 antagonistic fragments.

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Cathepsin K is responsible for cleaving ANGPT2 into Tie2 antagonistic fr...
(A) RAW264.7 cells pretreated with vehicle or increasing concentrations of CATK inhibitor odanacatib (ODN, from left 12.5 nM, 125 nM, 1.25 μM, 12.5 μM). IC50 = 108 nM for mouse Catk (59). RAW264.7 cells were stimulated with LPS and then incubated with recombinant ANGPT2. ODN was added to the cells 1 hour prior to LPS and ANGPT2. (B) Incubation of recombinant ANGPT2 and CM-MqLPS harvested from CRISPR-mediated Catk-KO RAW264.7 clonal cell lines and isogenic Cas9 control cell lines. CRISPR-mediated targeting strategy and Catk-KO scores of the 3 cell lines are described in Supplemental Figure 4. (C) Incubation of recombinant CATK and conditioned media of HEK293 cells transfected with wild-type or mutant Angpt2 expression vectors at predicted Catk cleavage sites for cANGPT250, for cANGPT225, or for both sites (Double). (D) Left: Western analysis under reduced conditions (10% β-mercaptoethanol) of CM-MqLPS versus CM-MqVeh incubated with recombinant ANGPT2. Arrows indicate cANGPT250 (orange) and cANGPT225 (violet). Right: Same samples as shown to the left applied to SDS-PAGE followed by Western analysis under nonreducing conditions. Predicted oligomeric status is indicated. Following LPS treatment, the 150 kDa band may indicate heterotetramerization of 2 cANGPT250 and 2 cANGPT225 monomers. (E) Western analysis of phospho-AKT and total AKT in HUVECs stimulated with different recombinant versions of cleaved ANGPT2 protein (1,000 ng/mL). All samples were loaded on the same gel but were noncontiguous. (F) Western analysis of phospho-AKT and total AKT in HUVECs stimulated with ANGPT1 (80 ng/mL) and CM of control empty vector (EV) or cANGPT2-expressing HEK293 cells. Western analysis images are representative of at least 3 independent experiments.