S-nitrosylation is required for ß2AR desensitization and experimental asthma.

The ß2-adrenergic receptor (ß2AR), a prototypic G-protein-coupled receptor (GPCR), is a powerful driver of bronchorelaxation, but the effectiveness of ß-agonist drugs in asthma is limited by desensitization and tachyphylaxis. We find that during activation, the ß2AR is modified by S-nitrosylation, which is essential for both classic desensitization by PKA as well as desensitization of NO-based signaling that mediates bronchorelaxation. Strikingly, S-nitrosylation alone can drive ß2AR internalization in the absence of traditional agonist. Mutant ß2AR refractory to S-nitrosylation (Cys265Ser) exhibits reduced desensitization and internalization, thereby amplifying NO-based signaling, and mice with Cys265Ser mutation are resistant to bronchoconstriction, inflammation, and the development of asthma. S-nitrosylation is thus a central mechanism in ß2AR signaling that may be operative widely among GPCRs and targeted for therapeutic gain.

Predicting Extubation Readiness in Preterm Infants Utilizing Machine Learning: A Diagnostic Utility Study.

OBJECTIVE: The objective of this study was to predict extubation readiness in preterm infants using machine learning analysis of bedside pulse oximeter and ventilator data. STUDY DESIGN: This is an observational study with prospective recordings of oxygen saturation (SpO2) and ventilator data from infants <30 weeks of gestation age. Research pulse oximeters collected SpO2 (1 Hz sampling rate) to quantify intermittent hypoxemia (IH). Continuous ventilator metrics were collected (4-5-minute sampling) from bedside ventilators. Data modeling was completed using unbiased machine learning algorithms. Three model sets were created using the following data source combinations: (1) IH and ventilator (IH + SIMV), (2) IH, and (3) ventilator (SIMV). Infants were also analyzed separated by postnatal age (infants <2 or ≥2 weeks of age). Models were compared by area under the receiver operating characteristic curve (AUC). RESULTS: A total of 110 extubation events from 110 preterm infants were analyzed. Infants had a median gestation age and birth weight of 26 weeks and 825 g, respectively. Of the 3 models presented, the IH + SIMV model achieved the highest AUC of 0.77 for all infants. Separating infants by postnatal age increased accuracy further achieving AUC of 0.94 for <2 weeks of age group and AUC of 0.83 for ≥2 weeks group. CONCLUSIONS: Machine learning analysis has the potential to enhance prediction accuracy of extubation readiness in preterm infants while utilizing readily available data streams from bedside pulse oximeters and ventilators.

Postnatal corticosteroid therapy in bronchopulmonary dysplasia - why animal studies disagree with clinical trials?

The systematic review and meta-analysis of newborn animal models by Irene Lok et al. is the first to extensively summarize the literature regarding postnatal systemic corticosteroid use on lung development of newborn rodent models. The meta-analysis showed that the use of postnatal corticosteroids resulted in a reduction in body weight along with persistent alveolar simplification. The most frequently used corticosteroid was dexamethasone. Corticosteroids have been extensively used in clinical trials in preterm newborns. Trials using early systemic administration of corticosteroids reduced the rate of BPD or mortality with no increase in the rates of cerebral palsy. Use of late systemic corticosteroids (administered >7 days after birth) also reduced the rate of BPD, mortality, and combined outcome of mortality or BPD. Late systemic corticosteroids showed no impact on the rates of neurodevelopmental outcomes in later childhood. It is important to note that later stages of inflammation leading to a more severe form of BPD continues to be a problem with no clear therapy in sight. The authors made a critical point in their paper - the negative effects of steroids were greater in the normal lung control animals than in the injured. This conveys caution in using steroids in a prophylactic manner. IMPACT: Use of systemic corticosteroids in clinical trials have shown good response in preterm neonates evidenced by reduced rate of bronchopulmonary dysplasia. Rodent models have not shown a similar beneficial response. Use of systemic corticosteroids have caused greater arrest of lung development in rodent models with normal lungs compared to those with lung damage.

Hypoxemia events in preterm neonates are associated with urine oxidative biomarkers.

BACKGROUND: Intermittent hypoxemia (IH) events are common in preterm neonates and are associated with adverse outcomes. Animal IH models can induce oxidative stress. We hypothesized that an association exists between IH and elevated peroxidation products in preterm neonates. METHODS: Time in hypoxemia, frequency of IH, and duration of IH events were assessed from a prospective cohort of 170 neonates (<31 weeks gestation). Urine was collected at 1 week and 1 month. Samples were analyzed for lipid, protein, and DNA oxidation biomarkers. RESULTS: At 1 week, adjusted multiple quantile regression showed positive associations between several hypoxemia parameters with various individual quantiles of isofurans, neurofurans, dihomo-isoprostanes, dihomo-isofurans, and ortho-tyrosine and a negative correlation with dihomo-isoprostanes and meta-tyrosine. At 1 month, positive associations were found between several hypoxemia parameters with quantiles of isoprostanes, dihomo-isoprostanes and dihomo-isofurans and a negative correlation with isoprostanes, isofurans, neuroprostanes, and meta-tyrosine. CONCLUSIONS: Preterm neonates experience oxidative damage to lipids, proteins, and DNA that can be analyzed from urine samples. Our single-center data suggest that specific markers of oxidative stress may be related to IH exposure. Future studies are needed to better understand mechanisms and relationships to morbidities of prematurity. IMPACT: Hypoxemia events are frequent in preterm infants and are associated with poor outcomes. The mechanisms by which hypoxemia events result in adverse neural and respiratory outcomes may include oxidative stress to lipids, proteins, and DNA. This study begins to explore associations between hypoxemia parameters and products of oxidative stress in preterm infants. Oxidative stress biomarkers may assist in identifying high-risk neonates.

Bronchopulmonary Dysplasia and Pulmonary Hypertension. The Role of Smooth Muscle adh5.

Bronchopulmonary dysplasia (BPD) is characterized by alveolar simplification, airway hyperreactivity, and pulmonary hypertension. In our BPD model, we have investigated the metabolism of the bronchodilator and pulmonary vasodilator GSNO (S-nitrosoglutathione). We have shown the GSNO catabolic enzyme encoded by adh5 (alcohol dehydrogenase-5), GSNO reductase, is epigenetically upregulated in hyperoxia. Here, we investigated the distribution of GSNO reductase expression in human BPD and created an animal model that recapitulates the human data. Blinded comparisons of GSNO reductase protein expression were performed in human lung tissues from infants and children with and without BPD. BPD phenotypes were evaluated in global (adh5-/-) and conditional smooth muscle (smooth muscle/adh5-/-) adh5 knockout mice. GSNO reductase was prominently expressed in the airways and vessels of human BPD subjects. Compared with controls, expression was greater in BPD smooth muscle, particularly in vascular smooth muscle (2.4-fold; P = 0.003). The BPD mouse model of neonatal hyperoxia caused significant alveolar simplification, airway hyperreactivity, and right ventricular and vessel hypertrophy. Global adh5-/- mice were protected from all three aspects of BPD, whereas smooth muscle/adh5-/- mice were only protected from pulmonary hypertensive changes. These data suggest adh5 is required for the development of BPD. Expression in the pulmonary vasculature is relevant to the pathophysiology of BPD-associated pulmonary hypertension. GSNO-mimetic agents or GSNO reductase inhibitors, both of which are currently in clinical trials for other conditions, could be considered for further study in BPD.

Neonatal intermittent hypoxemia events are associated with diagnosis of bronchopulmonary dysplasia at 36 weeks postmenstrual age.

BACKGROUND: Bronchopulmonary dysplasia (BPD) is a chronic lung disease and major pulmonary complication after premature birth. We have previously shown that increased intermittent hypoxemia (IH) events have been correlated to adverse outcomes and mortality in extremely premature infants. We hypothesize that early IH patterns are associated with the development of BPD. METHODS: IH frequency, duration, and nadirs were assessed using oxygen saturation (SpO2) waveforms in a retrospective cohort of 137 extremely premature newborns (<28 weeks gestation). Daily levels of inspired oxygen and mean airway pressure exposures were also recorded. RESULTS: Diagnosis of BPD at 36 weeks postmenstrual age was associated with increased daily IH, longer IH duration, and a higher IH nadir. Significant differences were detected through day 7 to day 26 of life. Infants who developed BPD had lower mean SpO2 despite their exposure to increased inspired oxygen and increased mean airway pressure. CONCLUSIONS: BPD was associated with more frequent, longer, and less severe IH events in addition to increased oxygen and pressure exposure within the first 26 days of life. Early IH patterns may contribute to the development of BPD or aid in identification of neonates at high risk.

Long-term effects of recurrent intermittent hypoxia and hyperoxia on respiratory system mechanics in neonatal mice.

BACKGROUND: Premature infants are at increased risk for wheezing disorders. Clinically, these neonates experience recurrent episodes of apnea and desaturation often treated by increasing the fraction of inspired oxygen (FIO2). We developed a novel paradigm of neonatal intermittent hypoxia with subsequent hyperoxia overshoots (CIHO/E) and hypothesized that CIHO/E elicits long-term changes on pulmonary mechanics in mice. METHODS: Neonatal C57BL/6 mice received CIHO/E, which consisted of 10% O2 (1 min) followed by a transient exposure to 50% FIO2, on 10-min repeating cycles 24 h/d from birth to P7. Baseline respiratory mechanics, methacholine challenge, RT-PCR for pro and antioxidants, radial alveolar counts, and airway smooth muscle actin were assessed at P21 after 2-wk room air recovery. Control groups were mice exposed to normoxia, chronic intermittent hyperoxia (CIHE), and chronic intermittent hypoxia (CIHO). RESULTS: CIHO/E and CIHE increased airway resistance at higher doses of methacholine and decreased baseline compliance compared with normoxia mice. Lung mRNA for NOX2 was increased by CIHO/E. Radial alveolar counts and airway smooth muscle actin was not different between groups. CONCLUSION: Neonatal intermittent hypoxia/hyperoxia exposure results in long-term changes in respiratory mechanics. We speculate that recurrent desaturation with hyperoxia overshoot may increase oxidative stress and contribute to wheezing in former preterm infants.

S-Nitrosoglutathione Attenuates Airway Hyperresponsiveness in Murine Bronchopulmonary Dysplasia.

Bronchopulmonary dysplasia (BPD) is characterized by lifelong obstructive lung disease and profound, refractory bronchospasm. It is observed among survivors of premature birth who have been treated with prolonged supplemental oxygen. Therapeutic options are limited. Using a neonatal mouse model of BPD, we show that hyperoxia increases activity and expression of a mediator of endogenous bronchoconstriction, S-nitrosoglutathione (GSNO) reductase. MicroRNA-342-3p, predicted in silico and shown in this study in vitro to suppress expression of GSNO reductase, was decreased in hyperoxia-exposed pups. Both pretreatment with aerosolized GSNO and inhibition of GSNO reductase attenuated airway hyperresponsiveness in vivo among juvenile and adult mice exposed to neonatal hyperoxia. Our data suggest that neonatal hyperoxia exposure causes detrimental effects on airway hyperreactivity through microRNA-342-3p-mediated upregulation of GSNO reductase expression. Furthermore, our data demonstrate that this adverse effect can be overcome by supplementing its substrate, GSNO, or by inhibiting the enzyme itself. Rates of BPD have not improved over the past two decades; nor have new therapies been developed. GSNO-based therapies are a novel treatment of the respiratory problems that patients with BPD experience.

Association between Intermittent Hypoxemia and NICU Length of Stay in Preterm Infants.

INTRODUCTION: Length of hospitalization varies widely in preterm infants and can be affected by multiple maternal and neonatal factors including respiratory instability. Therefore, we aimed to determine the association between postnatal intermittent hypoxemia (IH) and prolonged hospitalization. METHODS: This prospective single-center cohort study followed infants born at <31 weeks of gestational age through 2 years corrected age with detailed oxygen saturation data captured from days 7 to 30 of age. RESULTS: 51/164 (31%) of infants were discharged after 400/7 weeks of corrected gestational age (CGA). A greater average daily number of IH events (OR per 10 events/day 1.33 [95% CI 1.03-1.72]), duration of events (OR per minute 1.14 [1.07-1.21]), and percent time with oxygen saturation <80% (OR per percent 1.88 [1.25-2.85]) on days 7-30 of age were all significantly associated with prolonged hospitalization past 400/7 weeks CGA. In survival analyses, infants with a greater average daily number of IH events (HR per 10 events/day 0.89 [0.81-0.98]), percent time with oxygen saturation <80% (HR per percent 0.79 [0.67-0.94]), and duration of events (HR per minute 0.93 [0.91-0.95]) on days 7-30 of age all had significantly lower probability of earlier discharge. In addition, there was a significant interaction with gestational age; the association between IH and prolonged hospitalization was stronger in more mature infants (p = 0.024). CONCLUSIONS: Physiological instability on days 7-30 of age, as manifested by IH, is significantly associated with prolonged hospitalization. IH likely represents both a marker of initial severity of illness and the beginning of biological cascades, leading to prematurity-associated morbidities.

Rho-kinase inhibitors protect against neonatal hyperoxia-induced airway hyperreactivity in a rat pup model: Role of prostaglandin F2α.

BACKGROUND: Oxygen therapy in preterm neonates is associated with airway hyperreactivity. The role of Rho/Rho-kinase smooth muscle signaling in hyperoxia-induced airway hyperreactivity remains understudied. We hypothesized that inhibition of Rho-kinase will attenuate airway hyperreactivity induced by neonatal hyperoxia. METHODS: Newborn rats were raised in hyperoxia (>95% O2 ) or ambient air (AA) for 7 days. Subgroups were injected with a Rho-kinase inhibitor: Y-27632 (10 mg·kg-1 ·day-1 ) or fasudil (10 mg·kg-1 ·day-1 ), or a FP receptor antagonist - AS604872 (30 mg·kg-1 ·day-1 ). After exposures, tracheal cylinders were prepared for in vitro wire myography. Contraction to methacholine or PGF2α was measured in the presence or absence of tissue-bath Y-27632, fasudil, or AS604872. Lung PGF2α levels, Rho-kinase protein level and Rho-kinase 1 activity were measured by ELISA. RESULTS: Tracheal smooth muscle contraction was significantly greater in hyperoxic compared to AA groups. Both, Y-27632 and fasudil significantly decreased contractility to MCh or PGF2α in hyperoxic groups versus hyperoxic controls (p < 0.001), but did not alter AA group responses. Inhibition of FP receptors attenuated responses to PGF2α . Hyperoxia significantly increased lung PGF2α compared to AA (p < 0.01), but Rho-kinase inhibition did not influence PGF2α level. Rho-kinase protein level (p < 0.001) and activity (p < 0.01), were increased by hyperoxia, but blockade of FP receptor reduced the Rho-kinase 1 activity (p < 0.05) under hyperoxic condition. CONCLUSIONS: This study demonstrates an active role of Rho/Rho-kinase signaling on hyperoxia-induced airway hyperreactivity. These findings suggest that Rho-kinase inhibitors might serve as an effective therapy for hyperoxia-induced airway hyperreactivity.

NOX4 Mediates Epithelial Cell Death in Hyperoxic Acute Lung Injury Through Mitochondrial Reactive Oxygen Species.

Management of acute respiratory distress involves O2 supplementation, which is lifesaving, but causes severe hyperoxic acute lung injury (HALI). NADPH oxidase (NOX) could be a major source of reactive oxygen species (ROS) in hyperoxia (HO). Epithelial cell death is a crucial step in the development of many lung diseases. Alveolar type II (AT2) cells are the metabolically active epithelial cells of alveoli that serve as a source of AT1 cells following lung injury. The aim of this study was to determine the possible role of AT2 epithelial cell NOX4 in epithelial cell death from HALI. Wild type (WT), Nox4 fl/fl (control), and Nox4 -/- Spc-Cre mice were exposed to room air (NO) or 95% O2 (HO) to investigate the structural and functional changes in the lung. C57BL/6J WT animals subjected to HO showed increased expression of lung NOX4 compared to NO. Significant HALI, increased bronchoalveolar lavage cell counts, increased protein levels, elevated proinflammatory cytokines and increased AT2 cell death seen in hyperoxic Nox4 fl/fl control mice were attenuated in HO-exposed Nox4 -/- Spc-Cre mice. HO-induced expression of NOX4 in MLE cells resulted in increased mitochondrial (mt) superoxide production and cell apoptosis, which was reduced in NOX4 siRNA silenced cells. This study demonstrates a novel role for epithelial cell NOX4 in accelerating lung epithelial cell apoptosis from HALI. Deletion of the Nox4 gene in AT2 cells or silencing NOX4 in lung epithelial cells protected the lungs from severe HALI with reduced apoptosis and decreased mt ROS production in HO. These results suggest NOX4 as a potential target for the treatment of HALI.

The relationship between intermittent hypoxemia events and neural outcomes in neonates.

This brief review examines 1) patterns of intermittent hypoxemia in extremely preterm infants during early postnatal life, 2) the relationship between neonatal intermittent hypoxemia exposure and outcomes in both human and animal models, 3) potential mechanistic pathways, and 4) future alterations in clinical care that may reduce morbidity. Intermittent hypoxemia events are pervasive in extremely preterm infants (<28 weeks gestation at birth) during early postnatal life. An increased frequency of intermittent hypoxemia events has been associated with a range of poor neural outcomes including language and cognitive delays, motor impairment, retinopathy of prematurity, impaired control of breathing, and intraventricular hemorrhage. Neonatal rodent models have shown that exposure to short repetitive cycles of hypoxia induce a pathophysiological cascade. However, not all patterns of intermittent hypoxia are deleterious and some may even improve neurodevelopmental outcomes. Therapeutic interventions include supplemental oxygen, pressure support and pharmacologic drugs but prolonged hyperoxia and pressure exposure have been associated with cardiopulmonary morbidity. Therefore, it becomes imperative to distinguish high risk from neutral and/or even beneficial patterns of intermittent hypoxemia during early postnatal life. Identification of such patterns could improve clinical care with targeted interventions for high-risk patterns and minimal or no exposure to treatment modalities for low-risk patterns.

Neonatal Respiratory Distress Secondary to Meconium Aspiration Syndrome.

Infants born through meconium-stained amniotic fluid (MSAF) are 100 times more likely than infants born through clear amniotic fluid to develop respiratory distress in the neonatal period. Meconium aspiration syndrome (MAS) is a common cause of respiratory distress in term and post-mature neonates. MAS is defined as respiratory distress accompanied by a supplemental oxygen requirement in an infant born with MSAF, in the absence of any other identified etiology to explain the symptoms. Therapy for MAS is supportive, and should be tailored to each infant's specific pathophysiology. In cases of MAS with severe persistent pulmonary hypertension of the newborn (PPHN), patients may remain hypoxic despite aggressive ventilation, and in these cases surfactant, inhaled nitric oxide (iNO) and extracorporeal membrane oxygenation (ECMO) can be life-saving. Long-term prognosis for MAS is more related to severity of initial hypoxemia and possible neurological insult than to the pulmonary pathology.

Aerosolized Surfactant for Preterm Infants with Respiratory Distress Syndrome.

Currently, the administration of surfactant to preterm infants with respiratory distress syndrome (RDS) mainly relies on intratracheal instillation; however, there is increasing evidence of aerosolized surfactant being an effective non-invasive strategy. We present a historical narrative spanning sixty years of development of aerosolization systems. We also offer an overview of the pertinent mechanisms needed to create and manage the ideal aerosolization system, with a focus on delivery, distribution, deposition, and dispersion in the context of the human lung. More studies are needed to optimize treatment with aerosolized surfactants, including determination of ideal dosages, nebulizer types, non-invasive interfaces, and breath synchronization. However, the field is rapidly evolving, and widespread clinical use may be achieved in the near future.

Extubation Readiness in Preterm Infants: Evaluating the Role of Monitoring Intermittent Hypoxemia.

Preterm infants with respiratory distress may require mechanical ventilation which is associated with increased pulmonary morbidities. Prompt and successful extubation to noninvasive support is a pressing goal. In this communication, we show original data that increased recurring intermittent hypoxemia (IH, oxygen saturation <80%) may be associated with extubation failure at 72 h in a cohort of neonates <30 weeks gestational age. Current-generation bedside high-resolution pulse oximeters provide saturation profiles that may be of use in identifying extubation readiness and failure. A larger prospective study that utilizes intermittent hypoxemia as an adjunct predictor for extubation readiness is warranted.

Response to first dose of inhaled albuterol in mechanically ventilated preterm infants.

BACKGROUND: Bronchodilator responses among preterm infants are heterogeneous. Bedside measurements may identify responders. STUDY DESIGN: Respiratory measurements (Resistance, Compliance, FiO2) and pulse oximetry (SpO2) patterns were downloaded from infants <30 weeks gestational age during the first 2 months of life. Mechanically ventilated infants who received albuterol were included (n = 33). Measurements were compared before and after first albuterol. Secondary analyses assessed subsequent doses. RESULTS: Median gestation and birthweight were 25 3/7 weeks and 730 g, respectively. Mean Resistance decreased post-albuterol (p = 0.007). Sixty-eight percent of infants were responders based on decreased Resistance. Compliance and FiO2 did not significantly differ. Percent time in hypoxemia (SpO2 < 85%) decreased post albuterol (p < 0.02). In responders, Resistance changes diminished with subsequent administration (all p = 0.01). CONCLUSIONS: Ventilator resistance decreased in two-thirds of preterm infants, consistent with studies that utilized formal pulmonary function testing. Albuterol had a variable effect on delivered FiO2; however, hypoxemia may be useful in evaluating albuterol response.

Prenatal Maternal Lipopolysaccharide and Mild Newborn Hyperoxia Increase Intrapulmonary Airway but Not Vessel Reactivity in a Mouse Model.

Maternal infection is a risk for preterm delivery. Preterm newborns often require supplemental oxygen to treat neonatal respiratory distress. Newborn hyperoxia exposure is associated with airway and vascular hyperreactivity, while the complications of maternal infection are variable. In a mouse model of prenatal maternal intraperitoneal lipopolysaccharide (LPS, embryonic day 18) with subsequent newborn hyperoxia (40% oxygen × 7 days) precision-cut living lung slices were used to measure intrapulmonary airway and vascular reactivity at 21 days of age. Hyperoxia increased airway reactivity to methacholine compared to room air controls. Prenatal maternal LPS did not alter airway reactivity in room air. Combined maternal LPS and hyperoxia exposures increased airway reactivity vs. controls, although maximal responses were diminished compared to hyperoxia alone. Vessel reactivity to serotonin did not significantly differ in hyperoxia or room air; however, prenatal maternal LPS appeared to attenuate vessel reactivity in room air. Following room air recovery, LPS with hyperoxia lungs displayed upregulated inflammatory and fibrosis genes compared to room air saline controls (TNFαR1, iNOS, and TGFß). In this model, mild newborn hyperoxia increases airway but not vessel reactivity. Prenatal maternal LPS did not further increase hyperoxic airway reactivity. However, inflammatory genes remain upregulated weeks after recovery from maternal LPS and newborn hyperoxia exposures.