Abstract-In the pulmonary vasculature, cGMP concentrations are regulated in part by a cGMP-dependent phosphodiesterase (PDE), PDE5. Infants with persistent pulmonary hypertension of the newborn (PPHN) are often mechanically ventilated with high oxygen concentrations. The effects of hyperoxia on the developing pulmonary vasculature and PDE5 are largely unknown. Here, we demonstrate that exposure of fetal pulmonary artery smooth muscle cells (FPASMCs) to high levels of oxygen for 24 hours leads to decreased responsiveness to exogenous NO, as determined by a decreased intracellular cGMP response, increased PDE5 mRNA and protein expression, as well as increased PDE5 cGMP hydrolytic activity. We demonstrate that inhibition of PDE5 activity with sildenafil partially rescues cGMP responsiveness to exogenous NO. In FPASMCs, hyperoxia leads to increased oxidative stress without increasing cell death. Treatment of normoxic FPASMCs with H 2 O 2 is sufficient to induce PDE5 expression and activity, suggesting that reactive oxygen species mediate the effects of hyperoxia in FPASMCs. In support of this mechanism, a chemical antioxidant, N-acetyl-cysteine, is sufficient to block the hyperoxia-mediated increase in PDE5 expression and activity and rescue cGMP responsiveness to exogenous NO. Finally, ventilation of healthy neonatal sheep with 100% O 2 for 24 hours leads to increased PDE5 protein expression in the resistance pulmonary arteries and increased PDE5 activity in whole lung extracts. These data suggest that PDE5 expression and activity play a critical role in modulating neonatal pulmonary vascular tone in response to common clinical treatments for PPHN, such as oxygen and inhaled NO. Key Words: pulmonary circulation Ⅲ persistent pulmonary hypertension of the newborn Ⅲ cyclic GMP Ⅲ phosphodiesterases P ersistent pulmonary hypertension of the newborn (PPHN) is a clinical syndrome that results from a failure of the pulmonary vasculature to transition to extrauterine life. Infants typically present shortly after birth with respiratory distress and cyanosis but a structurally normal heart. The estimated incidence of PPHN is 0.2% of liveborn term infants. 1 PPHN is a manifestation of a heterogeneous group of disorders, and the response to therapy frequently depends on the underlying disease. Despite advances in clinical therapy, including high frequency ventilation, inhaled nitric oxide (iNO), and extracorporeal membrane oxygenation, there is still significant morbidity and mortality associated with this disease. Furthermore, iNO does not improve survival, and many infants do not respond or sustain their response to iNO for reasons that are unclear. 2,3 One factor that confounds the treatment of these infants is the use of supplemental oxygen. Oxygen has long been considered a vasodilator in the pulmonary circulation, and thus, 100% O 2 is considered a first-line therapy in infants with PPHN. 1,4 However, data from the adult acute respiratory distress syndrome literature, 5 from the neonatal bronchopulmonary dysplasia lite...

Rationale: Chronic hypoxia induces pulmonary vascular remodeling, pulmonary hypertension, and right ventricular hypertrophy. At present, little is known about mechanisms driving these responses. Hypoxia-inducible factor-1a (HIF-1a) is a master regulator of transcription in hypoxic cells, up-regulating genes involved in energy metabolism, proliferation, and extracellular matrix reorganization. Systemic loss of a single HIF-1a allele has been shown to attenuate hypoxic pulmonary hypertension, but the cells contributing to this response have not been identified.Objectives: We sought to determine the contribution of HIF-1a in smooth muscle on pulmonary vascular and right heart responses to chronic hypoxia.Methods: We used mice with homozygous conditional deletion of HIF-1a combined with tamoxifen-inducible smooth muscle-specific Cre recombinase expression. Mice received either tamoxifen or vehicle followed by exposure to either normoxia or chronic hypoxia (10% O 2 ) for 30 days before measurement of cardiopulmonary responses.Measurements and Main Results: Tamoxifen-induced smooth muscle-specific deletion of HIF-1a attenuated pulmonary vascular remodeling and pulmonary hypertension in chronic hypoxia. However, right ventricular hypertrophy was unchanged despite attenuated pulmonary pressures.Conclusions: These results indicate that HIF-1a in smooth muscle contributes to pulmonary vascular remodeling and pulmonary hypertension in chronic hypoxia. However, loss of HIF-1 function in smooth muscle does not affect hypoxic cardiac remodeling, suggesting that the cardiac hypertrophy response is not directly coupled to the increase in pulmonary artery pressure.

Endothelial nitric oxide (NO) synthase (eNOS) expression and activity are decreased in fetal lambs with persistent pulmonary hypertension (PPHN). We sought to determine the impact of mechanical ventilation with O2 with or without inhaled NO (iNO) or recombinant human SOD (rhSOD) on eNOS in the ductal ligation model of PPHN. PPHN lambs and age-matched controls were ventilated with 100% O2 for 24 h alone or combined with 20 ppm iNO continuously or a single dose of rhSOD (5 mg/kg) given intratracheally at delivery. In 1-day spontaneously breathing lambs, eNOS expression in resistance pulmonary arteries increased relative to fetal levels. eNOS expression increased in control lambs ventilated with 100% O2, but not in PPHN lambs. Addition of iNO or rhSOD increased eNOS expression and decreased generation of reactive oxygen species (ROS) in PPHN lambs relative to those ventilated with 100% O 2 alone. However, only rhSOD restored eNOS function, increased tetrahydrobiopterin (BH4), a critical cofactor for eNOS function, and restored GTP cyclohydrolase I expression in isolated vessels and lungs from PPHN lambs. These data suggest that ventilation of PPHN lambs with 100% O2 increases ROS production, blunts postnatal increases in eNOS expression, and decreases available BH4 in PPHN lambs. Although the addition of iNO or rhSOD diminished ROS production and increased eNOS expression, only rhSOD improved eNOS function and levels of available BH4. Thus therapies designed to decrease oxidative stress and restore eNOS coupling, such as rhSOD, may prove useful in the treatment of PPHN in newborn infants.reactive oxygen species; biopterin AS PART OF THE NORMAL PHYSIOLOGICAL transition at birth, the pulmonary vascular resistance decreases through complex pathways allowing pulmonary blood flow to increase by 10-fold. Physical and biochemical processes that contribute to the normal newborn pulmonary transition include mechanical distension of the lungs and increased PO 2 , which stimulate endothelial nitric oxide (NO) synthase (eNOS) (28,34,40). eNOS converts L-arginine to L-citrulline and NO, which in turn activates soluble guanylate cyclase in vascular smooth muscle cells to generate cGMP, ultimately leading to vasodilation (1). Emerging evidence continues to increase the understanding of the complex regulation of eNOS expression and activity, including the potential effects of O 2 (12, 33).Persistent pulmonary hypertension of the newborn (PPHN) is a clinical syndrome occurring in 2-6 per 1,000 live births, with a significant risk of death, as well as short-and long-term morbidity (19,44). It is caused by multiple disease processes, which lead to an abnormal transition at birth, resulting in continued elevated pulmonary vascular resistance, right-to left-sided extrapulmonary shunting of deoxygenated blood, and hypoxemia. Pathological findings include pulmonary vascular remodeling and smooth muscle hyperplasia in the absence of significant lung parenchyma pathology (15,29), changes that may be the result of prolonged fetal stress and...

Phosphodiesterase 5 (PDE5) and soluble guanylate cyclase (sGC) are key regulators of cGMP and pulmonary vascular tone. We sought to determine the impact of mechanical ventilation with O(2) with or without inhaled nitric oxide (iNO) or recombinant human Cu/Zn SOD (rhSOD) on sGC, PDE5, and cGMP in the ovine ductal ligation model of persistent pulmonary hypertension of the newborn (PPHN). PPHN lambs were ventilated with 100% O(2) for 24 h alone or combined with either inhalation of 20 parts per million (ppm) iNO continuously or a single intratracheal dose of rhSOD (5 mg/kg). Ventilated PPHN lambs were compared with PPHN fetuses, control fetuses, and 1-day-old spontaneously breathing lambs (1DSB). In the small pulmonary arteries of 1DSB lambs, sGC expression increased, PDE5 expression decreased, and cGMP concentrations increased relative to fetal levels. In PPHN lambs ventilated with 100% O(2), sGC activity increased to levels comparable with 1DSB levels. However, PDE5 expression and activity increased, and cGMP levels remained at fetal levels. Addition of either iNO or rhSOD decreased PDE5 expression and activity in PPHN lambs and increased cGMP levels to levels comparable with 1DSB lambs. These data suggest that ventilation of PPHN lambs with 100% O(2) impairs cGMP-mediated vasodilation in part due to increased PDE5 expression and activity. The addition of either iNO or rhSOD normalized PDE5 and cGMP levels. Thus therapies designed to decrease PDE5 and increase cGMP, such as iNO and rhSOD, may prove useful in the treatment of PPHN in newborn infants.