Validation of a human angiopoietin-2 ELISA for measurement of angiopoietin-2 concentrations in canine plasma samples and supernatant of primary canine aortic endothelial cell cultures.

Abstract

OBJECTIVE To assess 2 human ELISA kits for measurement of angiopoietin-1 and -2 concentrations in canine plasma samples, determine whether plasma angiopoeitin-2 concentration differed between septic and healthy dogs, and determine the effect of tumor necrosis factor-α (TNF-α) stimulation on angiopoeitin-2 release from primary canine aortic endothelial cells (pCAECs) in vitro. ANIMALS 10 healthy dogs and 10 septic dogs. PROCEDURES Human angiopoietin-1 and -2 ELISAs were used to detect recombinant canine angiopoietins-1 and -2 in canine plasma samples. The angiopoietin-2 ELISA was further validated by use of plasma samples from healthy and septic dogs and supernatants of pCAEC cultures. Associations between plasma angiopoeitin-2 and C-reactive protein (CRP) concentrations were examined. RESULTS Angiopoeitin-2 but not angiopoeitin-1 was detected in canine plasma samples by the respective ELISAs. The angiopoeitin-2 ELISA had excellent dilutional linearity, parallelism, accuracy, precision, and reproducibility for measurements in canine plasma samples and pCAEC supernatants. Plasma angiopoeitin-2 concentration was significantly higher in septic dogs (median, 25.5 ng/mL) than in healthy dogs (median, 6.7 ng/mL) and was positively correlated with plasma CRP concentration (R2 = 0.60). Stimulation of pCAECs with TNF-α resulted in a significant increase in supernatant angiopoietin-2 concentration. CONCLUSIONS AND CLINICAL RELEVANCE The tested human angiopoietin-2 ELISA kit was useful for measuring angiopoietin-2 concentrations in canine plasma samples and pCAEC supernatants. Sepsis appeared to increase angiopoietin-2 concentration in dogs in vivo, whereas TNF-α stimulation caused release of angiopoietin-2 from pCAECs in vitro. These findings support the use of angiopoietin-2 as a marker of endothelial cell activation and inflammation in dogs.