(1561) Cleavage by Cathepsin K of Angiopoietin-2 Creates Tie2 Antagonist Fragments in Sepsis

Introduction: Elevated Angiopoietin-2 (ANGPT2) is associated with diverse inflammatory conditions including sepsis, a leading global cause of mortality. During inflammation, ANGPT2 antagonizes the endothelium-enriched receptor Tie2 to destabilize the vasculature. In other contexts, ANGPT2 stimulates Tie2. The basis for context-dependent antagonism remains incompletely understood. Here we show that inflammation-induced proteolytic cleavage of ANGPT2 converts this ligand from Tie2 agonist to antagonist.

Methods: Vehicle- or ODN- pretreated RAW264.7 cells were stimulated with LPS and incubated with recombinant ANGPT2. For mouse survival experiment, hydrodynamic gene transfer of different plasmids (10ugDNA/BW); vehicle or ODN (20mg/kg, I.P.) was administered 1 hour prior to LPS (10mg/kg)(*p < 0.05). The empty vector (EV)+ODN group served as a control. For clinical data, we measured cANGPT2-25kDa (ng/mL) in sera of healthy controls (healthy, n=10), patients in the intensive care unit (ICU) with a primary diagnosis of acute respiratory distress syndrome (ARDS, n=20), ICU patients with a primary diagnosis of ARDS and sepsis (ARDS & Sepsis, n=10) and ICU patients with a primary diagnosis of sepsis (Sepsis, n=10)(**p < 0.01, ***p < 0.001).

Results: Conditioned media from stimulated macrophages induced ANGPT2 secretion. Unexpectedly, this was associated with reduction of the 75kDa full-length protein and appearance of new 25 and 50kDa 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 50kDa ANGPT2 fragments (cANGPT2-25, cANGPT2-50) bound and antagonized Tie2. Cathepsin K inhibition with the Phase-3 small molecule inhibitor odanacatib improved survival in distinct murine sepsis models. Odanacatib’s benefit was reversed by heterologous cANGPT2-25kDa. Full-length ANGPT2 enhanced survival in endotoxemic mice administered odanacatib and conversely increased mortality in the drug’s absence. Septic humans accumulated circulating ANGPT2 fragments, which were associated with adverse outcomes.

Conclusions: These results identify a novel proteolytic mechanism for the conversion of ANGPT2 from Tie2 agonist to antagonist with therapeutic implications for inflammatory conditions associated with ANGPT2 induction.