Objective. Inhaled nitric oxide (iNO) reduces the use of extracorporeal membrane oxygenation (ECMO)/incidence of death in term and near-term neonates with severe hypoxic respiratory failure. We conducted a randomized, double masked, multicenter trial to determine whether administration of iNO earlier in respiratory failure results in additional reduction in the incidence of these outcomes. Methods. Neonates who were born at ≥34 weeks’ gestation were enrolled when they required assisted ventilation and had an oxygenation index (OI) ≥15 and <25 on any 2 measurements in a 12-hour interval. Infants were randomized to early iNO or to simulated initiation of iNO (control). Infants who had an increase in OI to 25 or more were given iNO as standard therapy. Results. The trial enrollment was halted after 75% of target sample size was reached because of decreasing availability of eligible patients. The 150 infants who were given early iNO and 149 control infants had similar baseline characteristics. Arterial oxygen tension increased by >20 mm Hg in 73% of early iNO and 37% of control infants after study gas initiation. Control infants received standard iNO and deteriorated to OI >40 more often than infants who were given early iNO. The incidence of death (early iNO, 6.7% vs control, 9.4%), ECMO (10.7% vs 12.1%), and their combined incidence (16.7% vs 19.5%) were similar in both groups. Conclusion. iNO improves oxygenation but does not reduce the incidence of ECMO/mortality when initiated at an OI of 15 to 25 compared with initiation at >25 in term and near-term neonates with respiratory failure.

Persistent pulmonary hypertension of newborn (PPHN) is associated with decreased NO release and impaired pulmonary vasodilation. We investigated the hypothesis that increased superoxide (O₂^•−) release by an uncoupled endothelial nitric oxide synthase (eNOS) contributes to impaired pulmonary vasodilation in PPHN. We investigated the response of isolated pulmonary arteries to the NOS agonist ATP and the NO donor S-nitroso- N-acetylpenicillamine (SNAP) in fetal lambs with PPHN induced by prenatal ligation of ductus arteriosus and in sham-ligated controls in the presence or absence of the NOS antagonist nitro-l-arginine methyl ester (l-NAME) or the O₂^•−scavenger 4,5-dihydroxy-1,3-benzenedisulfonate (Tiron). ATP caused dose-dependent relaxation of pulmonary artery rings in control lambs but induced constriction of the rings in PPHN lambs. l-NAME, the NO precursor l-arginine, and Tiron restored the relaxation response of pulmonary artery rings to ATP in PPHN. Relaxation to NO was attenuated in arteries from PPHN lambs, and the response was improved by l-NAME and by Tiron. We also investigated the alteration in heat shock protein (HSP)90-eNOS interactions and release of NO and O₂^•−in response to ATP in the pulmonary artery endothelial cells (PAEC) from these lambs. Cultured PAEC and endothelium of freshly isolated pulmonary arteries from PPHN lambs released O₂^•−in response to ATP, and this was attenuated by the NOS antagonist l-NAME and superoxide dismutase (SOD). ATP stimulated HSP90-eNOS interactions in PAEC from control but not PPHN lambs. HSP90 immunoprecipitated from PPHN pulmonary arteries had increased nitrotyrosine signal. Oxidant stress from uncoupled eNOS contributes to impaired pulmonary vasodilation in PPHN induced by ductal ligation in fetal lambs.

Persistent pulmonary hypertension of newborn (PPHN) is associated with decreased nitric oxide (NO) release and impaired pulmonary vasodilation. We investigated the hypothesis that decreased association of heat shock protein 90 (HSP90) with endothelial NO synthase (eNOS) impairs NO release and vasodilation in PPHN. The responses to the NOS agonist ATP were investigated in fetal lambs with PPHN induced by prenatal ligation of ductus arteriosus, and in sham ligation controls. ATP caused dose-dependent vasodilation in control pulmonary resistance arteries, and this response was attenuated in PPHN vessels. The response of control pulmonary arteries to ATP was attenuated by N^G-nitro-l-arginine methyl ester (l-NAME), a NOS antagonist, and geldanamycin, an inhibitor of HSP90-eNOS interaction. The attenuated response to ATP observed in PPHN was improved by pretreatment of vessels with l-NAME or 4,5-dihydroxy-1,3-benzene-disulfonate, a superoxide scavenger. Pulmonary arteries from PPHN lambs had decreased basal levels of HSP90 in association with eNOS. Association of HSP90 with eNOS and NO release increased in response to ATP in control pulmonary artery endothelial cells, but not in cells from PPHN lambs. Decreased HSP90-eNOS interactions may contribute to the impaired NO release and vasodilation observed in the ductal ligation model of PPHN.

Pulmonary hypertension (PH) is a devastating pulmonary vascular disease characterized by aberrant muscularization of the normally nonmuscularized distal pulmonary arterioles. The expression of the transcription factor, Twist1, increases in the lungs of patients with pulmonary arterial hypertension. However, the mechanisms by which Twist1 controls the pathogenesis of PH remain unclear. It is becoming clear that endothelial-to-mesenchymal transition (EndMT) contributes to various vascular pathologies, including PH; Twist1 is known to mediate EndMT. In this report, we demonstrate that Twist1 overexpression increases transforming growth factor (TGF) β receptor2 (TGF-βR2) expression and Smad2 phosphorylation, and induces EndMT in cultured human pulmonary arterial endothelial (HPAE) cells, whereas a mutant construct of Twist1 at the serine 42 residue (Twist1S42A) fails to induce EndMT. We also implanted fibrin gel supplemented with HPAE cells on the mouse lung, and found that these HPAE cells form vascular structures and that Twist1-overexpressing HPAE cells undergo EndMT in the gel, whereas Twist1S42A-overexpressing cells do not. Furthermore, hypoxia-induced EndMT is inhibited in endothelial cells overexpressing Twist1S42A mutant construct in vitro. Hypoxia-induced accumulation of α-smooth muscle actin–positive cells in the pulmonary arterioles is attenuated in Tie2-specific Twist1 conditional knockout mice in vivo. These findings suggest that Twist1 serine 42 phosphorylation plays a key role in EndMT through TGF-β signaling and that modulation of Twist1 phosphorylation could be an effective strategy for managing PH.