Apocynin improves oxygenation and increases eNOS in persistent pulmonary hypertension of the newborn

Abstract

RH. Apocynin improves oxygenation and increases eNOS in persistent pulmonary hypertension of the newborn. Am J Physiol Lung Cell Mol Physiol 302: L616 -L626, 2012. First published December 23, 2011 doi:10.1152/ajplung.00064.2011.-NADPH oxidase is a major source of superoxide anions in the pulmonary arteries (PA). We previously reported that intratracheal SOD improves oxygenation and restores endothelial nitric oxide (NO) synthase (eNOS) function in lambs with persistent pulmonary hypertension of the newborn (PPHN). In this study, we determined the effects of the NADPH oxidase inhibitor apocynin on oxygenation, reactive oxygen species (ROS) levels, and NO signaling in PPHN lambs. PPHN was induced in lambs by antenatal ligation of the ductus arteriosus 9 days prior to delivery. Lambs were treated with vehicle or apocynin (3 mg/kg intratracheally) at birth and then ventilated with 100% O2 for 24 h. A significant improvement in oxygenation was observed in apocynin-treated lambs after 24 h of ventilation. Contractility of isolated fifth-generation PA to norepinephrine was attenuated in apocynin-treated lambs. PA constrictions to NO synthase (NOS) inhibition with N-nitro-L-arginine were blunted in PPHN lambs; apocynin restored contractility to N-nitro-L-arginine, suggesting increased NOS activity. Intratracheal apocynin also enhanced PA relaxations to the eNOS activator A-23187 and to the NO donor S-nitrosyl-N-acetyl-penicillamine. Apocynin decreased the interaction between NADPH oxidase subunits p22 phox and p47 phox and decreased the expression of Nox2 and p22 phox in ventilated PPHN lungs. These findings were associated with decreased superoxide and 3-nitrotyrosine levels in the PA of apocynin-treated PPHN lambs. eNOS protein expression, endothelial NO levels, and tetrahydrobiopterin-to-dihydrobiopterin ratios were significantly increased in PA from apocynin-treated lambs, although cGMP levels did not significantly increase and phosphodiesterase-5 activity did not significantly decrease. NADPH oxidase inhibition with apocynin may improve oxygenation, in part, by attenuating ROS-mediated vasoconstriction and by increasing NOS activity. reactive oxygen species; NADPH oxidase inhibition; nitric oxide signaling WHEN THE PULMONARY CIRCULATION fails to respond to various stimuli at birth, such as increased PO 2 and ventilation, it does not undergo the shift from the high-resistance, low-flow state in utero to the low-resistance, high-flow system postnatally (16,25). This failure results in persistent pulmonary hypertension of the newborn (PPHN) and is associated with impaired pulmonary gas exchange and oxygenation. Clinical strategies, such as mechanical ventilation with high concentrations of O 2 to correct hypoxemia and inhaled nitric oxide (NO), are commonly used to promote pulmonary vasodilation, but sustained improvement is not observed in ϳ40% of patients with PPHN (21). Hyperoxic ventilation also promotes formation of reactive oxygen species (ROS), such as superoxide anions, increases pulmonary artery (PA) contra...

Increased p22phox/Nox4 Expression Is Involved in Remodeling Through Hydrogen Peroxide Signaling in Experimental Persistent Pulmonary Hypertension of the Newborn

“…In the current study, we found an increase in expression of the superoxide generating isoform Nox2, but not Nox1, in PPHN PA and PASMC. We previously reported increased expression of the Nox2 activating protein p67 phox in fetal PPHN lambs relative to fetal controls (7), and we recently demonstrated that apocynin, an inhibitor of leukocyte Nox2 and a scavenger of H 2 O 2 in vitro (18), decreased p22 phox and p47 phox expression in ventilated PPHN lambs (46). While superoxide generated by Nox2 can contribute to elevated H 2 O 2 in PPHN, our siRNA data suggest that Nox4 is the major source of increased ROS in PPHN PASMC.…”

“…Recent studies demonstrated the increased NFjB activity in small pulmonary arterial lesions and in macrophages from explanted lungs of patients with idiopathic pulmonary arterial hypertension (22), and that inhibition of NFjB via pharmacological inhibition or delivery of a nanoparticle-mediated NFjB decoy attenuated monocrotaline-induced pulmonary arterial hypertension in rats (22,35). We previously reported increased expression of other NFjB target genes in PPHN, including inducible nitric oxide synthase (14), which may contribute to elevated levels of peroxynitrite in PPHN (46) and increase vasoconstriction of the neonatal pulmonary circulation (4,23). We suggest that the Nox4-induced NFjB activity may be involved in several dysregulated pathways in PPHN, and could be an important potential therapeutic target in PPHN.…”

Cyclic stretch stimulates mitochondrial reactive oxygen species and Nox4 signaling in pulmonary artery smooth muscle cells

“…Although the underlying mechanisms are incompletely understood, they involve impaired nitric oxide (NO) synthase (NOS)-mediated vasodilation and increased levels of reactive oxygen species (ROS) (23,43). PPHN lambs also exhibit elevated pulmonary vascular oxidant stress relative to control lambs (7,50), and recent studies have demonstrated improved oxygenation in ventilated PPHN lambs that received intratracheal antioxidants at birth (13,49,50). ROS may promote vasoconstriction directly and by impairing NO-mediated vasodilation (6,13,39), suggesting that the sources of increased ROS generation in PPHN are potential therapeutic targets.…”

The Role of Genetic Polymorphisms in Antioxidant Enzymes and Potential Antioxidant Therapies in Neonatal Lung Disease

“…Finally, since NADPH oxidase was proved a major source of superoxide anions in pulmonary arteries in different models of PPHN (38, 60) and the treatment with NADPH oxidase inhibitor was proved effective in reducing ROS in pulmonary arteries of PPHN models (186), the associated therapy of rhSOD and NADPH oxidase may offer additive benefits and, therefore, deserves to be investigated for this purpose.…”