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Persistent pulmonary hypertension of newborn (PPHN) is associated with impaired pulmonary vasodilation at birth. Previous studies demonstrated that a decrease in angiogenesis contributes to this failure of postnatal adaptation. We investigated the hypothesis that oxidative stress from NADPH oxidase (Nox) contributes to impaired angiogenesis in PPHN. PPHN was induced in fetal lambs by ductus arteriosus ligation at 85% of term gestation. Pulmonary artery endothelial cells (PAEC) from fetal lambs with PPHN (HTFL-PAEC) or control lambs (NFL-PAEC) were compared for their angiogenic activities and superoxide production. HTFL-PAEC had decreased tube formation, cell proliferation, scratch recovery, and cell invasion and increased cell apoptosis. Superoxide (O(2)(-)) production, measured by dihydroethidium epifluorescence and HPLC, were increased in HTFL-PAEC compared with NFL-PAEC. The mRNA levels for Nox2, Rac1, p47(phox), and Nox4, protein levels of p67(phox) and Rac1, and NADPH oxidase activity were increased in HTFL-PAEC. NADPH oxidase inhibitor, apocynin (Apo), and antioxidant, N-acetyl-cysteine (NAC), improved angiogenic measures in HTFL-PAEC. Apo and NAC also reduced apoptosis in HTFL-PAEC. Our data suggest that PPHN is associated with increased O(2)(-) production from NADPH oxidase in PAEC. Increased oxidative stress from NADPH oxidase contributes to the impaired angiogenesis of PAEC in PPHN.

Increased superoxide production contributes to the impaired angiogenesis of fetal pulmonary arteries with in utero pulmonary hypertension