P0762 - Subtyping Meconium Protease Activities Which Degrade Lung Protective Angiotensin Converting Enzyme-2 in Human Alveolar Epithelial Cells.

Background: Meconium pneumonitis occurs due to local lung injury and inflammation in newborns with meconium aspiration.The activation of the Renin Angiotensin System (RAS) plays a critical role in lung injury. Angiotensin converting enzyme- 2 (ACE 2) functions as a negative regulator of the angiotensin system. ACE-2 converts 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. Chymostatin is a strong inhibitor of many proteases, specifically chymotrypsin-like serine proteinase and chymase. It also inhibits lysosomal cysteine proteinases such as cathepsins A,B,C,H and L. Leupeptin is a reversible competitive inhibitor for serine and thiol proteases such as plasmin, trypsin, papain, cathepsin B,H, L whereas Pefobloc (AEBSF-aminoethylbenzenesulfonyl fluoride hydrochloride) is an irreversible inhibitor of serine proteases, e.g. trypsin, chymotrypsin, plasmin, thrombin, elastase. Chymostatin has shown to have a protective effect in acute lung injury.

Objective: To begin characterizing ACE-2-degrading proteases in meconium through the use of different subtypes of protease inhibitors.
Design/

Methods: Alveolar epithelial A549 cells were exposed to F-12 medium, 2.5% meconium and 2.5% meconium with specific protease inhibitor (PI). Specific PIs used in the study includes- chymostatin, AEBSF(Pefobloc) and leupeptin. At the end of incubation, cell lysates were collected for ACE-2 immunoblotting and enzyme activity. β-actin was used to normalize the assay.

Results: Reduction of ACE-2 enzyme 100-115kDa bands by meconium was decreased when treated with chymostatin, but not with the other PIs. The t-test showed a significant difference ( p value = 0.01) when treated with chymostatin and increased ACE-2 by 1.5-fold.
Conclusion(s): These data suggests that the inhibition of chymotrypsin-like serine-type proteases, but not trypsin or elastase in meconium, can reduce the degradation of lung protective ACE-2 by proteolytic enzymes. Increasing ACE2 activity through serine protease inhibitors might therefore be a novel approach for the treatment of meconium-induced lung injury.