Subtyping meconium protease activities which degrade lung protective angiotensin converting enzyme-2 in human lung cells

Abstract

Purpose: Meconium pneumonitis occurs due to local lung injury and inflammation in newborn with meconium aspiration. The activation of Renin Angiotensin System (RAS) plays critical role in lung injury. Angiotensin converting enzyme-2 (ACE 2) functions as a negative regulator of the angiotensin system by converting pro-apoptotic Angiotensin II to anti-apoptotic Angiotensin 1-7. Our previous study has shown that meconium causes degradation of lung protective ACE-2 by proteolytic enzymes present in meconium. However, the specific proteases in meconium that degrade ACE-2 have not yet been identified.  Objective: To begin characterizing ACE-2-degrading proteases in meconium through the use of different subtypes of protease inhibitors. Methods: Alveolar epithelial A549 cells were exposed to F-12 medium, 2.5% meconium and meconium + specific protease inhibitors (PIs). Specific PIs used included chymostatin, AEBSF(Pefobloc) and leupeptin. At the end of incubation, cell lysates were collected for ACE-2 immunoblotting and enzyme activity. Results: Reduction of ACE-2 immunoreactive 100-115 kDa bands or enzymatic activity by meconium was attenuated by treatment with chymostatin, but not with the other the PIs.  These data suggest the involvement of cysteine-like proteases in meconium in ACE-2 degradation, and suggest a potential therapeutic strategy of PI administration to babies aspirating meconium.