Journal: The Journal of Clinical Investigation
Article Title: Cathepsin K cleavage of angiopoietin-2 creates detrimental Tie2 antagonist fragments in sepsis
doi: 10.1172/JCI174135
Figure Lengend Snippet: ( A and B ) Western analysis of molecular weight column filtration (cut off 50 kDa) to separate full-length ANGPT2 and cANGPT2 25 . ( A ) His-tagged recombinant ANGPT2 was incubated with CM-Mq LPS and separated into a >50 kDa and a <50 kDa fraction. ANGPT2 and cANGPT2 25 were visualized with anti-His antibody. Fraction concentration factor was calculated by determining the weight of retentate (18.7 mg) and filtrate (75.1 mg) versus prefilter weight (99 mg). Representative blot of 3 independent experiments depicted, along with ( B ) quantified band signal intensities corrected by concentration factors and normalized to prefiltration band intensity set to 100% (mean ± SD, n = 3): >50 kDa ANGPT2, 97.27% ± 9.17%; >50 kDa cANGPT2 25 , 72.19% ± 5.40%; <50 kDa ANGPT2, 0%; <50 kDa cANGPT2 25, 20.37% ± 2.30%; and cANGPT2 25 detected in both fractions combined, 92.56% ± 7.49%). ( C ) cANGPT2 25 (ng/mL) in sera of healthy individuals acting as controls (healthy, n = 10), patients in the 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) (Kruskal-Wallis, **P < 0.01, ***P < 0.001). ( D ) cANGPT2 25 (ng/mL) stratified by survival ( n = 31) versus death ( n = 9) 28 days after diagnosis of ARDS, ARDS and sepsis, or sepsis (Mann Whitney, *P < 0.01). ( E ) Spearman’s correlation plot showing ICU patients with diagnoses of ARDS, ARDS and sepsis, or sepsis ( n = 40) comparing major parameters from blood tests and other assessments, as defined against absolute concentration of cANGPT2 25 (ng/mL) and the ratio of cANGPT2 25 to the total amount of ANGPT2. Color indicates strength of association by correlation coefficient. ICU LOS, ICU length of stay; Pmax, peak inspiratory pressure; PEEP, peak end-expiratory pressure; SOFA, sequential organ failure assessment score; INR, international normalized ratio measuring blood coagulation; PCT, procalcitonin; LDH, lactate dehydrogenase. ( F and G ) Correlation between cANGPT2 25 (ng/mL) in ICU patients and SOFA score ( F ) and total ANGPT2 (ng/mL). Data in G are from E . Triangles indicate death within 28 days of diagnosis.
Article Snippet: Recombinant TNF-α and mouse and human ANGPT2 were purchased from R&D Systems.
Techniques: Western Blot, Molecular Weight, Filtration, Recombinant, Incubation, Concentration Assay, Biomarker Discovery, MANN-WHITNEY, Coagulation