Inhaled Nitric Oxide at Birth Reduces Pulmonary Vascular Resistance and Improves Oxygenation in Preterm Lambs.

Resuscitation with 21% O2 may not achieve target oxygenation in preterm infants and in neonates with persistent pulmonary hypertension of the newborn (PPHN). Inhaled nitric oxide (iNO) at birth can reduce pulmonary vascular resistance (PVR) and improve PaO2. We studied the effect of iNO on oxygenation and changes in PVR in preterm lambs with and without PPHN during resuscitation and stabilization at birth. Preterm lambs with and without PPHN (induced by antenatal ductal ligation) were delivered at 134 d gestation (term is 147-150 d). Lambs without PPHN were ventilated with 21% O2, titrated O2 to maintain target oxygenation or 21% O2 + iNO (20 ppm) at birth for 30 min. Preterm lambs with PPHN were ventilated with 50% O2, titrated O2 or 50% O2 + iNO. Resuscitation with 21% O2 in preterm lambs and 50%O2 in PPHN lambs did not achieve target oxygenation. Inhaled NO significantly decreased PVR in all lambs and increased PaO2 in preterm lambs ventilated with 21% O2 similar to that achieved by titrated O2 (41 ± 9% at 30 min). Inhaled NO increased PaO2 to 45 ± 13, 45 ± 20 and 76 ± 11 mmHg with 50% O2, titrated O2 up to 100% and 50% O2 + iNO, respectively, in PPHN lambs. We concluded that iNO at birth reduces PVR and FiO2 required to achieve target PaO2.

Randomized trial of oxygen weaning strategies following chest compressions during neonatal resuscitation.

BACKGROUND: The Neonatal Resuscitation Program (NRP) recommends using 100% O2 during chest compressions and adjusting FiO2 based on SpO2 after return of spontaneous circulation (ROSC). The optimal strategy for adjusting FiO2 is not known. METHODS: Twenty-five near-term lambs asphyxiated by umbilical cord occlusion to cardiac arrest were resuscitated per NRP. Following ROSC, lambs were randomized to gradual decrease versus abrupt wean to 21% O2 followed by FiO2 titration to achieve NRP SpO2 targets. Carotid blood flow and blood gases were monitored. RESULTS: Three minutes after ROSC, PaO2 was 229 ± 32 mmHg in gradual wean group compared to 57 ± 13 following abrupt wean to 21% O2 (p < 0.001). PaO2 remained high in the gradual wean group at 10 min after ROSC (110 ± 10 vs. 67 ± 12, p < 0.01) despite similar FiO2 (~0.3) in both groups. Cerebral O2 delivery (C-DO2) was higher above physiological range following ROSC with gradual wean (p < 0.05). Lower blood oxidized/reduced glutathione ratio (suggesting less oxidative stress) was observed with abrupt wean. CONCLUSION: Weaning FiO2 abruptly to 0.21 with adjustment based on SpO2 prevents surge in PaO2 and C-DO2 and minimizes oxidative stress compared to gradual weaning from 100% O2 following ROSC. Clinical trials with neurodevelopmental outcomes comparing post-ROSC FiO2 weaning strategies are warranted. IMPACT: In a lamb model of perinatal asphyxial cardiac arrest, abrupt weaning of inspired oxygen to 21% prevents excessive oxygen delivery to the brain and oxidative stress compared to gradual weaning from 100% oxygen following return of spontaneous circulation. Clinical studies assessing neurodevelopmental outcomes comparing abrupt and gradual weaning of inspired oxygen after recovery from neonatal asphyxial arrest are warranted.

Malnutrition, poor post-natal growth, intestinal dysbiosis and the developing lung.

In extremely preterm infants, poor post-natal growth, intestinal dysbiosis and bronchopulmonary dysplasia are common, and each is associated with long-term complications. The central hypothesis that this review will address is that these three common conditions are interrelated. Challenges to studying this hypothesis include the understanding that malnutrition and poor post-natal growth are not synonymous and that there is not agreement on what constitutes a normal intestinal microbiota in this evolutionarily new population. If this hypothesis is supported, further study of whether "correcting" intestinal dysbiosis in extremely preterm infants reduces postnatal growth restriction and/or bronchopulmonary dysplasia is indicated.

Optimal Oxygen Targets in Term Lambs with Meconium Aspiration Syndrome and Pulmonary Hypertension.

Optimal oxygen saturation as measured by pulse oximetry (SpO2) in neonatal lung injury, such as meconium aspiration syndrome (MAS) and persistent pulmonary hypertension of newborn (PPHN), is not known. Our goal was to determine the SpO2 range in lambs with MAS and PPHN that results in the highest brain oxygen delivery (bDO2) and pulmonary blood flow (Qp) and the lowest pulmonary vascular resistance and oxidative stress. Meconium was instilled into endotracheal tubes in 25 near-term gestation lambs, and the umbilical cord was occluded to induce asphyxia and gasping, causing MAS and PPHN. Lambs were randomized into four groups and ventilated for 6 hours with fixed fraction of inspired oxygen (FiO2) = 1.0 irrespective of SpO2, and three groups had FiO2 titrated to keep preductal SpO2 between 85% and 89%, 90% and 94%, and 95% and 99%, respectively. Tissues were collected to measure nitric oxide synthase activity, 3-nitrotyrosine, and 8-isoprostanes. Throughout the 6-hour exposure period, lambs in the 95-99% SpO2 target group had the highest Qp, lowest pulmonary vascular resistance, and highest bDO2 but were exposed to higher FiO2 (0.5 ± 0.21 vs. 0.29 ± 0.17) with higher lung 3-nitrotyrosine (0.67 [interquartile range (IQR), 0.43-0.73] ng/mcg protein vs. 0.1 [IQR, 0.09-0.2] ng/mcg protein) and lower lung nitric oxide synthase activity (196 [IQR, 192-201] mMol nitrite/mg protein vs. 270 [IQR, 227-280] mMol nitrite/mg protein) compared with the 90-94% target group. Brain 3-nitrotyrosine was lower in the 85-89% target group, and brain/lung 8-isoprostane levels were not significantly different. In term lambs with MAS and PPHN, Qp and bDO2 through the first 6 hours are higher with target SpO2 in the 95-99% range. However, the 90-94% target range is associated with significantly lower FiO2 and lung oxidative stress. Clinical trials comparing the 90-94% versus the 95-99% SpO2 target range in term infants with PPHN are warranted.

Intestinal Dysbiosis and the Developing Lung: The Role of Toll-Like Receptor 4 in the Gut-Lung Axis.

Background: In extremely premature infants, postnatal growth restriction (PNGR) is common and increases the risk of developing bronchopulmonary dysplasia (BPD) and pulmonary hypertension (PH). Mechanisms by which poor nutrition impacts lung development are unknown, but alterations in the gut microbiota appear to play a role. In a rodent model, PNGR plus hyperoxia causes BPD and PH and increases intestinal Enterobacteriaceae, Gram-negative organisms that stimulate Toll-like receptor 4 (TLR4). We hypothesized that intestinal dysbiosis activates intestinal TLR4 triggering systemic inflammation which impacts lung development. Methods: Rat pups were assigned to litters of 17 (PNGR) or 10 (normal growth) at birth and exposed to room air or 75% oxygen for 14 days. Half of the pups were treated with the TLR4 inhibitor TAK-242 from birth or beginning at day 3. After 14 days, pulmonary arterial pressure was evaluated by echocardiography and hearts were examined for right ventricular hypertrophy (RVH). Lungs and serum samples were analyzed by western blotting and immunohistochemistry. Results: Postnatal growth restriction + hyperoxia increased pulmonary arterial pressure and RVH with trends toward increased plasma IL1ß and decreased IκBα, the inhibitor of NFκB, in lung tissue. Treatment with the TLR4 inhibitor attenuated PH and inflammation. Conclusion: Postnatal growth restriction induces an increase in intestinal Enterobacteriaceae leading to PH. Activation of the TLR4 pathway is a promising mechanism by which intestinal dysbiosis impacts the developing lung.

The developing gut-lung axis: postnatal growth restriction, intestinal dysbiosis, and pulmonary hypertension in a rodent model.

BACKGROUND: Postnatal growth restriction (PNGR) in premature infants increases risk of pulmonary hypertension (PH). In a rodent model, PNGR causes PH, while combining PNGR and hyperoxia increases PH severity. We hypothesized that PNGR causes intestinal dysbiosis and that treatment with a probiotic attenuates PNGR-associated PH. METHOD: Pups were randomized at birth to room air or 75% oxygen (hyperoxia), to normal milk intake (10 pups/dam) or PNGR (17 pups/dam), and to probiotic Lactobacillus reuteri DSM 17938 or phosphate-buffered saline. After 14 days, PH was assessed by echocardiography and right ventricular hypertrophy (RVH) was assessed by Fulton's index (right ventricular weight/left ventricle + septal weight). The small bowel and cecum were analyzed by high-throughput 16S ribosomal RNA gene sequencing. RESULTS: PNGR with or without hyperoxia (but not hyperoxia alone) altered the microbiota of the distal small bowel and cecum. Treatment with DSM 17938 attenuated PH and RVH in pups with PNGR, but not hyperoxia alone. DSM 17938 treatment decreased α-diversity. The intestinal microbiota differed based on oxygen exposure, litter size, and probiotic treatment. CONCLUSION: PNGR causes intestinal dysbiosis and PH. Treatment with DSM 17938 prevents PNGR-associated RVH and PH. Changes in the developing intestine and intestinal microbiota impact the developing lung vasculature and RV.

Optimal oxygenation and role of free radicals in PPHN.

Effective ventilation of the lungs is essential in mediating pulmonary vasodilation at birth to allow effective gas exchange and an increase in systemic oxygenation. Unsuccessful transition prevents the increase in pulmonary blood flow after birth resulting in hypoxemia and persistent pulmonary hypertension of the newborn (PPHN). Management of neonates with PPHN includes ventilation of the lungs with supplemental oxygen to correct hypoxemia. Optimal oxygenation should meet oxygen demand to the tissues and avoid hypoxic pulmonary vasoconstriction (HPV) while preventing oxidative stress. The optimal target for oxygenation in PPHN is not known. Animal models have demonstrated that PaO2<45 mmHg exacerbates HPV. However, there are no practical methods of assessing oxygen levels associated with oxidant stress. Oxidant stress can be due to free radical generation from underlying lung disease or from free radicals generated by supplemental oxygen. Free radicals act on the nitric oxide pathway reducing cGMP and promoting pulmonary vasoconstriction. Antioxidant therapy improves systemic oxygenation in an animal model of PPHN but there are no clinical trials to support such therapy. Targeting preductal SpO2 between 90 and 97% and PaO2 at 50-80 mmHg appears prudent in PPHN but clinical trials to support this practice are lacking. Preterm infants with PPHN present unique challenges due to lack of antioxidant defenses and functional and structural immaturity of the lungs. This review highlights the need for additional studies to mitigate the impact of oxidative stress in the lung and pulmonary vasculature in PPHN.

Somatic growth and the risks of bronchopulmonary dysplasia and pulmonary hypertension: connecting epidemiology and physiology 1.

In the premature infant, poor growth in utero (fetal growth restriction) and in the first weeks of life (postnatal growth restriction) are associated with increased risk for bronchopulmonary dysplasia and pulmonary hypertension. In this review, we summarize the epidemiologic data supporting these associations, present a novel rodent model of postnatal growth restriction, and review 5 promising mechanisms by which poor nutrition may affect the developing lung. These observations support the hypothesis that nutritional and (or) pharmacologic interventions early in life may be able to decrease risk of the pulmonary complications of extreme prematurity.

Umbilical cord blood metabolomics reveal distinct signatures of dyslipidemia prior to bronchopulmonary dysplasia and pulmonary hypertension.

Pulmonary hypertension (PH) is a common consequence of bronchopulmonary dysplasia (BPD) and remains a primary contributor to increased morbidity and mortality among preterm infants. Unfortunately, at the present time, there are no reliable early predictive markers for BPD-associated PH. Considering its health consequences, understanding in utero perturbations that lead to the development of BPD and BPD-associated PH and identifying early predictive markers is of utmost importance. As part of the discovery phase, we applied a multiplatform metabolomics approach consisting of untargeted and targeted methodologies to screen for metabolic perturbations in umbilical cord blood (UCB) plasma from preterm infants that did ( n = 21; cases) or did not ( n = 21; controls) develop subsequent PH. A total of 1,656 features were detected, of which 407 were annotated by metabolite structures. PH-associated metabolic perturbations were characterized by reductions in major choline-containing phospholipids, such as phosphatidylcholines and sphingomyelins, indicating altered lipid metabolism. The reduction in UCB abundances of major choline-containing phospholipids was confirmed in an independent validation cohort consisting of UCB plasmas from 10 cases and 10 controls matched for gestational age and BPD status. Subanalyses in the discovery cohort indicated that elevations in the oxylipins PGE1, PGE2, PGF2a, 9- and 13-HOTE, 9- and 13-HODE, and 9- and 13-KODE were positively associated with BPD presence and severity. This expansive evaluation of cord blood plasma identifies compounds reflecting dyslipidemia and suggests altered metabolite provision associated with metabolic immaturity that differentiate subjects, both by BPD severity and PH development.

Postnatal growth restriction augments oxygen-induced pulmonary hypertension in a neonatal rat model of bronchopulmonary dysplasia.

BACKGROUND: Prematurity and fetal growth restriction are risk factors for pulmonary hypertension (PH) in infants with bronchopulmonary dysplasia (BPD). Neonatal rats develop PH and vascular remodeling when exposed to hyperoxia. We hypothesize that postnatal growth restriction (PNGR) due to under-nutrition increases the severity of PH induced by hyperoxia in neonatal rats. METHODS: Pups were randomized at birth to litters maintained in room air or 75% oxygen (hyperoxia), together with litters of normal milk intake (10 pups) or PNGR (17 pups). After 14 d, right ventricular hypertrophy (RVH) was assessed by Fulton's index (right ventricular weight/left ventricular plus septal weight) and PH by echocardiography. Lungs were analyzed by immunohistochemistry, morphometrics, western blotting, and metabolomics. RESULTS: Hyperoxia and PNGR each significantly increased pulmonary arterial pressure, RVH and pulmonary arterial medial wall thickness, and significantly decreased pulmonary vessel number. These changes were significantly augmented in pups exposed to both insults. Hyperoxia and PNGR both significantly decreased expression of proteins involved in lung development and vasodilation. CONCLUSION: PNGR induces right ventricular and pulmonary vascular remodeling and augments the effects of oxygen in neonatal rats. This may be a powerful tool to investigate the mechanisms that induce PH in low-birth-weight preterm infants with BPD.

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

This study was designed to determine whether cyclic stretch induces a persistent pulmonary hypertension of the newborn (PPHN) phenotype of increased NADPH oxidase (Nox) 4 signaling in control pulmonary artery smooth muscle cells (PASMC), and to identify the signal transduction molecules involved. To achieve this, PPHN was induced in lambs by antenatal ligation of the ductus arteriosus at 128 days gestation. After 9 days, lungs and PASMC were isolated from control (twin) and PPHN lambs. Control PASMC were exposed to cyclic stretch at 1 Hz and 15% elongation for 24 h. Stretch-induced Nox4 expression was attenuated by inhibition of mitochondrial complex III and NF-κB, and stretch-induced protein thiol oxidation was attenuated by Nox4 small interfering RNA and complex III inhibition. NF-κB activity was increased by stretch in a complex III-dependent fashion, and stretch-induced cyclin D1 expression was attenuated by complex III inhibition and Nox4 small interfering RNA. This is the first study to show that cyclic stretch increases Nox4 expression via mitochondrial complex III-induced activation of NF-κB in fetal PASMC, resulting in ROS signaling and increased cyclin D1 expression. Targeting these signaling molecules may attenuate pulmonary vascular remodeling associated with PPHN.

Hypoxia inducible factor signaling and experimental persistent pulmonary hypertension of the newborn.

BACKGROUND: Mitochondrial reactive oxygen species (ROS) levels and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) activity are increased in a lamb model of persistent pulmonary hypertension of the newborn (PPHN). These events can trigger hypoxia inducible factor (HIF) signaling in response to hypoxia, which has been shown to contribute to pulmonary vascular remodeling in rodent models of pulmonary hypertension. However, the role of HIF signaling in chronic intrauterine pulmonary hypertension is not well understood. AIM: To determine if HIF signaling is increased in the lamb model of PPHN, and to identify the underlying mechanisms. RESULTS: PPHN was induced in lambs by antenatal ligation of the ductus arteriosus at 128 days gestation. After 9 days, lungs and pulmonary artery smooth muscle cells (PASMC) were isolated from control and PPHN lambs. HIF-1α expression was increased in PPHN lungs and HIF activity was increased in PPHN PASMC relative to controls. Hypoxia increased HIF activity to a greater degree in PPHN vs. control PASMC. Control PASMC were exposed to cyclic stretch at 1 Hz and 15% elongation for 24 h, as an in vitro model of vascular stress. Stretch increased HIF activity, which was attenuated by inhibition of mitochondrial complex III and NFκB. CONCLUSION: Increased HIF signaling in PPHN is triggered by stretch, via mechanisms involving mitochondrial ROS and NFκB. Hypoxia substantially amplifies HIF activity in PPHN vascular cells. Targeting these signaling molecules may attenuate and reverse pulmonary vascular remodeling associated with PPHN.

Hydrocortisone normalizes phosphodiesterase-5 activity in pulmonary artery smooth muscle cells from lambs with persistent pulmonary hypertension of the newborn.

Phosphodiesterase-5 (PDE5) is the primary phosphodiesterase in the pulmonary vasculature. It degrades cyclic guanosine monophosphate (cGMP) and inhibits cGMP-mediated vasorelaxation. We previously reported that hydrocortisone treatment decreased hyperoxia-induced PDE5 activity and markers of oxidative stress in lambs with persistent pulmonary hypertension of the newborn (PPHN) ventilated with 100% O2. The objective of our study was to determine the molecular mechanism by which hydrocortisone downregulates PDE5 and oxidative stress in fetal pulmonary artery smooth muscle cells (FPASMCs) from PPHN lambs. PPHN FPASMC were incubated for 24 hours in either 21% or 95% O2. Some cells were treated with 100 nM hydrocortisone and/or ±1 µM helenalin, an inhibitor of nuclear factor κ B (NFκB), a redox-sensitive transcription factor. Exposure to hyperoxia led to increased PDE5 activity, oxidative stress, and NFκB activity. Pretreatment of PPHN FPASMC with hydrocortisone normalized PDE5 activity, decreased cytosolic oxidative stress, increased expression of extracellular superoxide dismutase and NFκB inhibitory protein, and decreased NFκB activity. Similarly, treatment with NFκB inhibitor, helenalin, decreased PDE5 activity. These data suggest that hyperoxia activates NFκB, which in turn induces PDE5 activity in PPHN FPASMC, whereas treatment with hydrocortisone attenuates these changes by blocking reactive oxygen species-induced NFκB activity.

Role of reactive oxygen species in neonatal pulmonary vascular disease.

SIGNIFICANCE: Abnormal lung development in the perinatal period can result in severe neonatal complications, including persistent pulmonary hypertension (PH) of the newborn and bronchopulmonary dysplasia. Reactive oxygen species (ROS) play a substantive role in the development of PH associated with these diseases. ROS impair the normal pulmonary artery (PA) relaxation in response to vasodilators, and ROS are also implicated in pulmonary arterial remodeling, both of which can increase the severity of PH. RECENT ADVANCES: PA ROS levels are elevated when endogenous ROS-generating enzymes are activated and/or when endogenous ROS scavengers are inactivated. Animal models have provided valuable insights into ROS generators and scavengers that are dysregulated in different forms of neonatal PH, thus identifying potential therapeutic targets. CRITICAL ISSUES: General antioxidant therapy has proved ineffective in reversing PH, suggesting that it is necessary to target specific signaling pathways for successful therapy. FUTURE DIRECTIONS: Development of novel selective pharmacologic inhibitors along with nonantioxidant therapies may improve the treatment outcomes of patients with PH, while further investigation of the underlying mechanisms may enable earlier detection of the disease.

Developmental differences in hyperoxia-induced oxidative stress and cellular responses in the murine lung.

Exposure of newborn mice to high inspired oxygen elicits a distinct phenotype of compromised alveolar and vascular development, although lethality during long-term exposure is lower in newborns compared to adults. As the effects of hyperoxia are mediated by excessive reactive oxygen species (ROS) generation, we hypothesized that newborn mice may exhibit enhanced expression of antioxidant defenses or attenuated ROS generation compared with adults. We measured subcellular oxidant responses to acute hyperoxia in lung slices and alveolar epithelial cells at varying time points during postnatal murine lung development. Oxidant stress was assessed using RoGFP, a ratiometric protein thiol redox sensor, targeted to the cytosol or the mitochondrial matrix. In contrast to newborn resistance to oxygen-induced mortality, cells of lung slices from younger mice demonstrated exaggerated mitochondrial matrix oxidant stress compared to adults, whereas oxidant stress responses in the cytosol were absent. Cell death in lung slices from newborn mice exposed to 48h of hyperoxia was also greater than for adults. Consistent with these findings, expression of antioxidant enzymes in newborn lungs was lower than in adults, and induction of antioxidant levels and activity during 24h of in vivo exposure was absent. However, expression of the reactive oxygen species-generating enzyme NADPH oxidase 1 was increased with hyperoxic exposure in the young but not the adult lung. Collectively, these results suggest that the greater lethality in adult animals may be more likely attributed to processes such as inflammation than to differences in antioxidant defenses. Therapies for neonatal and adult oxidative lung injury should therefore consider and address developmental differences in oxidative stress responses.

Cyclic stretch induces inducible nitric oxide synthase and soluble guanylate cyclase in pulmonary artery smooth muscle cells.

In the pulmonary vasculature, mechanical forces such as cyclic stretch induce changes in vascular signaling, tone and remodeling. Nitric oxide is a potent regulator of soluble guanylate cyclase (sGC), which drives cGMP production, causing vasorelaxation. Pulmonary artery smooth muscle cells (PASMCs) express inducible nitric oxide synthase (iNOS), and while iNOS expression increases during late gestation, little is known about how cyclic stretch impacts this pathway. In this study, PASMC were subjected to cyclic stretch of 20% amplitude and frequency of 1 Hz for 24 h and compared to control cells maintained under static conditions. Cyclic stretch significantly increased cytosolic oxidative stress as compared to static cells (62.9 ± 5.9% vs. 33.3 ± 5.7% maximal oxidation), as measured by the intracellular redox sensor roGFP. Cyclic stretch also increased sGCß protein expression (2.5 ± 0.9-fold), sGC activity (1.5 ± 0.2-fold) and cGMP levels (1.8 ± 0.2-fold), as well as iNOS mRNA and protein expression (3.0 ± 0.9 and 2.6 ± 0.7-fold, respectively) relative to control cells. An antioxidant, recombinant human superoxide dismutase (rhSOD), significantly decreased stretch-induced cytosolic oxidative stress, but did not block stretch-induced sGC activity. Inhibition of iNOS with 1400 W or an iNOS-specific siRNA inhibited stretch-induced sGC activity by 30% and 68% respectively vs. static controls. In conclusion, cyclic stretch increases sGC expression and activity in an iNOS-dependent manner in PASMC from fetal lambs. The mechanism that produces iNOS and sGC upregulation is not yet known, but we speculate these effects represent an early compensatory mechanism to counteract the effects of stretch-induced oxidative stress. A better understanding of the interplay between these two distinct pathways could provide key insights into future avenues to treat infants with pulmonary hypertension.

Increased p22(phox)/Nox4 expression is involved in remodeling through hydrogen peroxide signaling in experimental persistent pulmonary hypertension of the newborn.

AIM: To determine if the NADPH oxidase isoform Nox4 contributes to increased H(2)O(2) generation in persistent pulmonary hypertension of the newborn (PPHN) pulmonary arteries (PA), and to identify downstream signaling targets of Nox4 that contribute to vascular remodeling and vasoconstriction. RESULTS: PPHN was induced in lambs by antenatal ligation of the ductus arteriosus at 128 days gestation. After 9 days, lungs, PA, and PA smooth muscle cells (PASMC) were isolated from control and PPHN lambs. Increased expression of p22(phox) and Nox4 in PPHN lungs, PA, and PASMC was associated with increased reactive oxygen species in PPHN PA, increased protein thiol oxidation in PPHN PASMC, and a decreased activity of extracellular superoxide dismutase (ecSOD) in the lungs and PASMC. Nox4 small interfering RNA (siRNA) decreased Nox4 expression and thiol oxidation and increased the ecSOD activity in PPHN PASMC. An increased activity of nuclear factor-kappa B (NFκB) and expression of its target gene cyclin D1 were detected in PPHN lungs, PA, and PASMC. Nox4 siRNA and catalase attenuated these increases in PASMC, and catalase decreased cyclin D1 expression in PPHN lungs. INNOVATION: This study demonstrates for the first time that Nox4 expression is elevated in a lamb model of neonatal pulmonary hypertension. It identifies increased NFκB and cyclin D1 expression and a decreased ecSOD activity as targets of increased Nox4 signaling. CONCLUSION: PPHN increases p22(phox) and Nox4 expression and activity resulting in elevated H(2)O(2) levels in PPHN PA. Increased H(2)O(2) induces vasoconstriction via mechanisms involving ecSOD inactivation, and stimulates vascular remodeling via NFκB activation and increased cyclin D1 expression. Approaches that inhibit the pulmonary arterial Nox4 activity may attenuate vasoconstriction and vascular remodeling in PPHN.

Brief hyperoxia increases mitochondrial oxidation and increases phosphodiesterase 5 activity in fetal pulmonary artery smooth muscle cells.

AIMS: Oxygen is a pulmonary vasodilator, but data suggest high O(2) concentrations impede that response. We previously reported 24 h of 100% O(2) increased phosphodiesterase 5 (PDE5) activity in fetal pulmonary artery smooth muscle cells (FPASMC) and in ventilated neonatal lambs. PDE5 degrades cyclic GMP (cGMP) and inhibits nitric oxide (NO)-mediated cGMP-dependent vasorelaxation. We sought to determine the mechanism by which hyperoxia initiates reactive oxygen species (ROS) production and regulates PDE5. RESULTS: Thirty minutes of hyperoxia increased mitochondrial ROS versus normoxia (30.3±1.7% vs. 21.1±2.8%), but had no effect on cytosolic ROS, measured by roGFP, a ratiometric protein thiol redox sensor. Hyperoxia increased PDE5 activity (220±39%) and decreased cGMP responsiveness to NO (37±17%). Mitochondrial catalase overexpression attenuated hyperoxia-induced mitochondrial roGFP oxidation, compared to FPASMC infected with empty adenoviral vector (50±3% of control) or mitochondrial superoxide dismutase. MitoTEMPO, mitochondrial catalase, and DT-3, a cGMP-dependent protein kinase I alpha inhibitor, decreased PDE5 activity (32±13%, 26±21%, and 63±10% of control, respectively), and restored cGMP responsiveness to NO (147±16%,172±29%, and 189±43% of control, respectively). C57Bl6 mice exposed to 90%-100% O(2) for 45 min±mechanical ventilation had increased PA PDE5 activity (206±39% and 235±75%, respectively). INNOVATION: This is the first description that hyperoxia induces ROS in the mitochondrial matrix prior to the cytosol. Our results indicate that short hyperoxia exposures can produce significant changes in critical cellular signaling pathways. CONCLUSIONS: These results indicate that mitochondrial matrix oxidant signals generated during hyperoxia, specifically H(2)O(2), activate PDE5 in a cGMP-dependent protein kinase-dependent manner in pulmonary vascular smooth muscle cells.

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

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% O(2) 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.

Hydrocortisone normalizes oxygenation and cGMP regulation in lambs with persistent pulmonary hypertension of the newborn.

In the pulmonary vasculature, cGMP levels are regulated by soluble guanylate cyclase (sGC) and phosphodiesterase 5 (PDE5). We previously reported that lambs with persistent pulmonary hypertension of the newborn (PPHN) demonstrate increased reactive oxygen species (ROS) and altered sGC and PDE5 activity, with resultant decreased cGMP. The objective of this study was to evaluate the effects of hydrocortisone on pulmonary vascular function, ROS, and cGMP in the ovine ductal ligation model of PPHN. PPHN lambs were ventilated with 100% O(2) for 24 h. Six lambs received 5 mg/kg hydrocortisone every 8 h times three doses (PPHN-hiHC), five lambs received 3 mg/kg hydrocortisone followed by 1 mg·kg(-1)·dose(-1) times two doses (PPHN-loHC), and six lambs were ventilated with O(2) alone (PPHN). All groups were compared with healthy 1-day spontaneously breathing lambs (1DSB). O(2) ventilation of PPHN lambs decreased sGC activity, increased PDE5 activity, and increased ROS vs. 1DSB lambs. Both hydrocortisone doses significantly improved arterial-to-alveolar ratios relative to PPHN lambs, decreased PDE5 activity, and increased cGMP relative to PPHN lambs. High-dose hydrocortisone also increased sGC activity, decreased PDE5 expression, decreased ROS, and increased total vascular SOD activity vs. PPHN lambs. These data suggest that hydrocortisone treatment in clinically relevant doses improves oxygenation and decreases hyperoxia-induced changes in sGC and PDE5 activity, increasing cGMP levels. Hydrocortisone reduces ROS levels in part by increasing SOD activity in PPHN lambs ventilated with 100% O(2.) We speculate that hydrocortisone increases cGMP by direct effects on sGC and PDE5 expression and by attenuating abnormalities induced by oxidant stress.