Abstract 18635: H2O2 and Peroxynitrite Mediate Extracellular Superoxide Dismutase Inactivation in Persistent Pulmonary Hypertension of the Newborn
Introduction: Inactivation of extracellular superoxide dismutase (ecSOD) may increase oxidant stress, prevent the normal decrease in pulmonary vascular resistance at birth, and contribute to persistent pulmonary hypertension (PPHN).
Hypothesis: Increased expression of NADPH oxidase and iNOS increase H2O2, superoxide, and peroxynitrite (ONOO) levels in PPHN, which lead to ecSOD inactivation.
Methods: PPHN was induced in lambs by antenatal ductal ligation at 128 days gestation. Pulmonary arterial smooth muscle cells (PASMC) were isolated from control and PPHN fetal lambs. Western blotting quantified gene expression and 3-nitrotyrosine (3-NT) protein levels as a marker of ONOO. Cell oxidation status was determined using the redox-sensitive probe roGFP. ecSOD protein was purified by affinity chromatography and SOD activity determined using a kit. Additional assays were performed in PPHN lambs ventilated with 100% oxygen for 24 hours with or without intratracheal catalase.
Results: PPHN PASMC displayed markedly increased oxidation of roGFP (3.7±0.4-fold) associated with increased expression of p22 (3.1±1.0-fold), Nox2 (2.1±0.4-fold), and Nox4 (2.0±0.5-fold). iNOS (1.7±0.3-fold) and 3-NT protein (2.1±0.3-fold) were increased relative to control PASMC, associated with increased ecSOD nitration (1.6±0.1-fold) and suppressed activity (0.4±0.2-fold). iNOS inhibition decreased nitrated ecSOD protein to control values, and increased ecSOD activity by 1.4±0.1-fold. H2O2 increased p22, Nox2 and Nox4 expression in control PASMC while catalase treatment increased ecSOD activity by 1.5±0.1-fold in PPHN PASMC. In lung tissue from ventilated PPHN lambs, we observed similar patterns of increased NADPH oxidase subunits, nitrated ecSOD, and decreased ecSOD activity relative to controls. Intratracheal catalase reduced expression of all three NADPH oxidase subunits, reduced nitrated ecSOD, restored ecSOD activity, and improved oxygenation.
Conclusion: H2O2 and ONOO inactivate pulmonary ecSOD, contributing to the vascular dysfunction of PPHN. Catalase scavenges H2O2, decreases p22, Nox2 and Nox4 expression, and reduces nitration of ecSOD, thus restoring activity and enhancing NO-mediated pulmonary artery vasodilation.
- © 2010 by American Heart Association, Inc.