Neonatal Hyperoxia Activates Activating Transcription Factor 4 to Stimulate Folate Metabolism and Alveolar Epithelial Type 2 Cell Proliferation.

Oxygen supplementation in preterm infants disrupts alveolar epithelial type 2 (AT2) cell proliferation through poorly understood mechanisms. Here, newborn mice are used to understand how hyperoxia stimulates an early aberrant wave of AT2 cell proliferation that occurs between Postnatal Days (PNDs) 0 and 4. RNA-sequencing analysis of AT2 cells isolated from PND4 mice revealed hyperoxia stimulates expression of mitochondrial-specific methylenetetrahydrofolate dehydrogenase 2 and other genes involved in mitochondrial one-carbon coupled folate metabolism and serine synthesis. The same genes are induced when AT2 cells normally proliferate on PND7 and when they proliferate in response to the mitogen fibroblast growth factor 7. However, hyperoxia selectively stimulated their expression via the stress-responsive activating transcription factor 4 (ATF4). Administration of the mitochondrial superoxide scavenger mitoTEMPO during hyperoxia suppressed ATF4 and thus early AT2 cell proliferation, but it had no effect on normative AT2 cell proliferation seen on PND7. Because ATF4 and methylenetetrahydrofolate dehydrogenase are detected in hyperplastic AT2 cells of preterm infant humans and baboons with bronchopulmonary dysplasia, dampening mitochondrial oxidative stress and ATF4 activation may provide new opportunities for controlling excess AT2 cell proliferation in neonatal lung disease.

Murine mechanical ventilation stimulates alveolar epithelial cell proliferation.

High tidal volume mechanical ventilation can cause inflammation and lung damage. Mechanical strain is also necessary for normal lung growth. The current work was performed to determine if mechanical ventilation with clinically utilized tidal volumes stimulates a proliferative response in the lung. Six- to 8-week-old C57/Bl6 mice, anesthetized with ketamine/xylozine, were ventilated for 6 hours with 10 mL/kg tidal volume, positive end-expiratory pressure (PEEP) 3cm H(2)O. Pulmonary function testing demonstrated decreased compliance within 3 hours of ventilation. Assessment of bronchoalveolar lavage (BAL) demonstrated no significant increase in lactate dehydrogenase, total lavagable cell number, or total protein after ventilation. There was evidence of inflammation in the lungs of ventilated mice, with an increased percentage of lymphocytes and neutrophils in BAL, and an increase in macrophage inflammatory protein (MIP)-2 and interleukin (IL)-1beta message in lung tissue. Immunohistochemistry of inflation-fixed lungs demonstrated increased alveolar cell proliferation, as measured by both proliferating cell nuclear antigen and Ki67 staining. Dual staining confirmed that proliferating cells labeled with proSP-B, demonstrating that ventilation induces proliferation of alveolar type II cells. Ventilation did not increase apoptosis in alveolar type II cells, as measured by TUNEL staining. Ventilation at low tidal volumes leads to a mild inflammatory response and alveolar epithelial cell proliferation.

Postnatal estradiol up-regulates lung nitric oxide synthases and improves lung function in bronchopulmonary dysplasia.

RATIONALE: Nitric oxide (NO) plays an important role in lung development and perinatal lung function, and pulmonary NO synthases (NOS) are decreased in bronchopulmonary dysplasia (BPD) following preterm birth. Fetal estradiol levels increase during late gestation and estradiol up-regulates NOS, suggesting that after preterm birth estradiol deprivation causes attenuated lung NOS resulting in impaired pulmonary function. OBJECTIVE: To test the effects of postnatal estradiol administration in a primate model of BPD over 14 days after delivery at 125 days of gestation (term = 185 d). METHODS: Cardiopulmonary function was assessed by echocardiography and whole body plethysmography. Lung morphometric and histopathologic analyses were performed, and NOS enzymatic activity and abundance were measured. MEASUREMENTS AND MAIN RESULTS: Estradiol caused an increase in blood pressure and ductus arteriosus closure. Expiratory resistance and lung compliance were also improved, and this occurred before spontaneous ductal closure. Furthermore, both oxygenation and ventilation indices were improved with estradiol, and the changes in lung function and ventilatory support requirements persisted throughout the study period. Whereas estradiol had negligible effect on indicators of lung inflammation and on lung structure assessed after the initial 14 days of ventilatory support, it caused an increase in lung neuronal and endothelial NOS enzymatic activity. CONCLUSIONS: In a primate model of BPD, postnatal estradiol treatment had favorable cardiovascular impact, enhanced pulmonary function, and lowered requirements for ventilatory support in association with an up-regulation of lung NOS. Estradiol may be an efficacious postnatal therapy to improve lung function and outcome in preterm infants.

Retinoids increase lung elastin expression but fail to alter morphology or angiogenesis genes in premature ventilated baboons.

Retinoids regulate elastin synthesis by alveolar myofibroblasts and affect angiogenesis pathways, both of which are processes critical for alveolar development. Retinoids accelerate alveolarization in rodents and are now used therapeutically in premature infants at risk of bronchopulmonary dysplasia (BPD). This study examined the effects of retinoid supplementation on alveolar elastin expression and deposition and angiogenesis-related signaling in a primate model of BPD. Premature baboons delivered at 125 d of gestation after maternal steroid treatment were given surfactant and ventilated with minimal supplemental oxygen for 14 d with (n = 5) and without (n = 5) supplemental vitamin A (5000 U/kg/d) and compared with 140-d unventilated controls. Ventilatory efficiency index (VEI) and oxygenation index (OI) were not statistically different between ventilated treatment groups. Expression of vascular endothelial growth factor A (VEGF-A), fms-related tyrosine kinase 1 (Flt-1), and tyrosine kinase with immunoglobulin-like and EGF-like domains 1 (TIE-1) was repressed by premature delivery and mechanical ventilation and was not altered by retinoid supplementation. Retinoid supplementation did not enhance alveolar angiogenesis. Elastin expression was repressed by premature delivery and extended ventilation, and retinoid supplementation increased elastin expression specifically in alveolar myofibroblasts within alveolar walls. These results suggest that the small decrease in mortality among premature infants receiving retinoid supplementation may not be mediated through enhanced alveolar development.

Pathogenesis of bronchopulmonary dysplasia.

Bronchopulmonary dysplasia (BPD), initially described 40 years ago, is a dynamic clinical entity that continues to affect tens of thousands of premature infants each year. BPD was first characterized as a fibrotic pulmonary endpoint following severe Respiratory Distress Syndrome (RDS). It was the result of pulmonary healing after RDS, high oxygen exposure, positive pressure ventilation, and poor bronchial drainage secondary to endotracheal intubation in premature infants. With improved treatment for RDS, including surfactant replacement, oxygen saturation monitoring, improved modes of mechanical ventilation, antibiotic therapies, nutritional support, and infants surviving at younger gestations, the clinical picture of BPD has changed. In the following pages, we will summarize the multifaceted pathophysiologic factors leading to the pulmonary changes in "new" BPD, which is primarily characterized by disordered or delayed development. The contribution of hyperoxia and hypoxia, mechanical forces, vascular maldevelopment, inflammation, fluid management, patent ductus arteriosus (PDA), nutrition, and genetics will be discussed.

Type II epithelial cells are critical target for hyperoxia-mediated impairment of postnatal lung development.

Type II epithelial cells are essential for lung development and remodeling, as they are precursors for type I cells and can produce vascular mitogens. Although type II cell proliferation takes place after hyperoxia, it is unclear why alveolar remodeling occurs normally in adults whereas it is permanently disrupted in newborns. Using a line of transgenic mice whose type II cells could be identified by their expression of enhanced green fluorescent protein and endogenous expression of surfactant proteins, we investigated the age-dependent effects of hyperoxia on type II cell proliferation and alveolar repair. In adult mice, type II cell proliferation was low during room air and hyperoxia exposure but increased during recovery in room air and then declined to control levels by day 7. Eight weeks later, type II cell number and alveolar compliance were indistinguishable from those in room air controls. In newborn mice, type II cell proliferation markedly increased between birth and postnatal day 7 before declining by postnatal day 14. Exposure to hyperoxia between postnatal days 1 and 4 inhibited type II cell proliferation, which resumed during recovery and was aberrantly elevated on postnatal day 14. Eight weeks later, recovered mice had 70% fewer type II cells and 30% increased lung compliance compared with control animals. Recovered mice also had higher levels of T1alpha, a protein expressed by type I cells, with minimal changes detected in genes expressed by vascular cells. These data suggest that perinatal hyperoxia adversely affects alveolar development by disrupting the proper timing of type II cell proliferation and differentiation into type I cells.

Hyperoxic ventilated premature baboons have increased p53, oxidant DNA damage and decreased VEGF expression.

Hyperoxia is implicated in the pathogenesis of bronchopulmonary dysplasia (BPD), a chronic lung disease of premature infants. High levels of supplemental oxygen can result in microvascular endothelial cell death and may disrupt lung development. In postnatal animals, hyperoxia inhibits expression of vascular endothelial growth factor (VEGF), which is required for normal vascular development. A potential mechanism of oxygen effects on VEGF is induction of p53, a transcription factor that represses VEGF gene transcription. Oxidant DNA damage can increase p53. We used a moderately premature baboon model of hyperoxia to examine p53, oxidant DNA damage, and VEGF expression. Fetal baboons delivered at 140 d of gestation (75% of term) were ventilated with 100% oxygen or oxygen as needed for 6 or 10 d. Lungs from the 10-d 100% oxygen animals had increased nuclear p53, compared with the oxygen as needed animals. The mechanism of increased p53 was probably related to oxidant DNA damage, which was documented by increased oxidized guanine. Dual fluorescent confocal microscopy found increased oxidized guanine in mitochondrial DNA of distal lung epithelial cells. Distal epithelial cell VEGF expression was decreased and p21, another downstream target of p53, was increased in the distal epithelium of the hyperoxic animals. These data show that p53 is induced in hyperoxic fetal lung epithelium and are consistent with p53 repression of VEGF expression in these cells. The findings suggest that oxidant DNA damage may be a mechanism of increased p53 in hyperoxic fetal lung.

Bronchopulmonary dysplasia in preterm infants: pathophysiology and management strategies.

Bronchopulmonary dysplasia (BPD) has classically been described as including inflammation, architectural disruption, fibrosis, and disordered/delayed development of the infant lung. As infants born at progressively earlier gestations have begun to survive the neonatal period, a 'new' BPD, consisting primarily of disordered/delayed development, has emerged. BPD causes not only significant complications in the newborn period, but is associated with continuing mortality, cardiopulmonary dysfunction, re-hospitalization, growth failure, and poor neurodevelopmental outcome after hospital discharge. Four major risk factors for BPD include premature birth, respiratory failure, oxygen supplementation, and mechanical ventilation, although it is unclear whether any of these factors is absolutely necessary for development of the condition. Genetic susceptibility, infection, and patent ductus arteriosus have also been implicated in the pathogenesis of the disease. The strategies with the strongest evidence for effectiveness in preventing or lessening the severity of BPD include prevention of prematurity and closure of a clinically significant patent ductus arteriosus. Some evidence of effectiveness also exists for single-course therapy with antenatal glucocorticoids in women at risk for delivering premature infants, surfactant replacement therapy in intubated infants with respiratory distress syndrome, retinol (vitamin A) therapy, and modes of respiratory support designed to minimize 'volutrauma' and oxygen toxicity. The most effective treatments for ameliorating symptoms or preventing exacerbation in established BPD include oxygen therapy, inhaled glucocorticoid therapy, and vaccination against respiratory pathogens.Many other strategies for the prevention or treatment of BPD have been proposed, but have weaker or conflicting evidence of effectiveness. In addition, many therapies have significant side effects, including the possibility of worsening the disease despite symptom improvement. For instance, supraphysiologic systemic doses of glucocorticoids lessen the incidence of BPD in infants at risk for the disease, and promote weaning of oxygen and mechanical ventilation in infants with established BPD. However, the side effects of systemic glucocorticoid therapy, most notably the recently recognized adverse effects on neurodevelopment, preclude their routine use for the prevention or treatment of BPD. Future research in BPD will most probably focus on continued incremental improvements in outcome, which are likely to be achieved through the combined effects of many therapeutic modalities.

In vivo exposure to hyperoxia induces DNA damage in a population of alveolar type II epithelial cells.

It is well established that hyperoxia injures and kills alveolar endothelial and type I epithelial cells of the lung. Although type II epithelial cells remain morphologically intact, it remains unclear whether they are also damaged. DNA integrity was investigated in adult mice whose type II cells were identified by their endogenous expression of pro-surfactant protein C or transgenic expression of enhanced green fluorescent protein. In mice exposed to room air, punctate perinuclear 8-oxoguanine staining was detected in approximately 4% of all alveolar cells and in 30% of type II cells. After 48 or 72 h of hyperoxia, 8-oxoguanine was detected in 11% of all alveolar cells and in >60% of type II cells. 8-Oxoguanine colocalized by confocal microscopy with the mitochondrial transmembrane protein cytochrome oxidase subunit 1. Type II cells isolated from hyperoxic lungs exhibited nuclear DNA strand breaks by comet assay even though they were viable and morphologically indistinguishable from cells isolated from lungs exposed to room air. These data reveal that type II cells exposed to in vivo hyperoxia have oxidized and fragmented DNA. Because type II cells are essential for lung remodeling, our findings raise the possibility that they are proficient in DNA repair.

Induced p21Cip1 in premature baboons with CLD: implications for alveolar hypoplasia.

Aberrant pulmonary epithelial and mesenchymal cell proliferation occurs when newborns are treated with oxygen and ventilation to mitigate chronic lung disease. Because the cyclin-dependent kinase inhibitor p21 inhibits proliferation of oxygen-exposed cells, its expression was investigated in premature baboons delivered at 125 days (67% of term) and treated with oxygen and ventilation pro re nata (PRN) for 2, 6, 14, and 21 days. Approximately 5% of all cells expressed p21 during normal lung development of which <1% of these cells were pro-surfactant protein (SP)-B-positive epithelial cells. The percentage of cells expressing p21 increased threefold in all PRN-treated animals, but different cell populations expressed it during disease progression. Between 2 and 6 days of treatment, p21 was detected in 30-40% of pro-SP-B cells. In contrast, only 12% of pro-SP-B cells expressed p21 by 14 and 21 days of treatment, by which time p21 was also detected in mesenchymal cells. Even though increased epithelial and mesenchymal cell proliferation occurs during disease progression, those cells expressing p21 did not also express the proliferative marker Ki67. Thus two populations of epithelial and mesenchymal cells can be identified that are either expressing Ki67 and proliferating or expressing p21 and not proliferating. These data suggest that p21 may play a role in disorganized proliferation and alveolar hypoplasia seen in newborn chronic lung disease.

Increased epithelial cell proliferation in very premature baboons with chronic lung disease.

Coordinated proliferation of lung cells is required for normal lung growth and differentiation. Chronic injury to developing lung may disrupt normal patterns of cell proliferation. To examine patterns of cell proliferation in injured developing lungs, we investigated premature baboons delivered at 125 days gestation (approximately 67% of term) and treated with oxygen and ventilation for 6, 14, or 21 days (PRN). Each PRN treatment group contained 3 or 4 animals. During normal in utero lung development, the proportion of proliferating lung cells declined as measured by the cell-cycle marker Ki67. In the PRN group, the proportion of proliferating lung cells was 2.5-8.5-fold greater than in corresponding gestational controls. By 14 days of treatment, the proportion of cells that expressed pro-surfactant protein B (proSP-B) was ~2.5-fold greater than in gestational controls. In the PRN group, 41% of proliferating cells expressed proSP-B compared with 5.8% in the gestational controls. By 21 days of treatment, proliferation of proSP-B-expressing epithelial cells declined substantially, but the proportion of proliferating non-proSP-B-expressing cells increased approximately sevenfold. These data show that the development of chronic lung disease is associated with major alterations in normal patterns of lung-cell proliferation.

Normal remodeling of the oxygen-injured lung requires the cyclin-dependent kinase inhibitor p21(Cip1/WAF1/Sdi1).

Alveolar cells of the lung are injured and killed when exposed to elevated levels of inspired oxygen. Damaged tissue architecture and pulmonary function is restored during recovery in room air as endothelial and type II epithelial cells proliferate. Although excessive fibroblast proliferation and inflammation occur when abnormal remodeling occurs, genes that regulate repair remain unknown. Our recent observation that hyperoxia inhibits proliferation through induction of the cyclin-dependent kinase inhibitor p21(Cip1/WAF1/Sdi1), which also facilitates DNA repair, suggested that p21 may participate in remodeling. This hypothesis was tested in p21-wild-type and -deficient mice exposed to 100% FiO(2) and recovered in room air. p21 increased during hyperoxia, remained elevated after 1 day of recovery before returning to unexposed levels. Increased proliferation occurred when p21 expression decreased. In contrast, higher and sustained levels of proliferation, resulting in myofibroblast hyperplasia and monocytic inflammation, occurred in recovered p21-deficient lungs. Cells with DNA strand breaks and expressing p53 were observed in hyperplastic regions suggesting that DNA integrity had not been restored. Normal recovery of endothelial and type II epithelial cells, as assessed by expression of cell-type-specific genes was also delayed in p21-deficient lungs. These results reveal that p21 is required for remodeling the oxygen-injured lung and suggest that failure to limit replication of damaged DNA may lead to cell death, inflammation, and abnormal remodeling. This observation has important implications for therapeutic strategies designed to attenuate long-term chronic lung disease after oxidant injury.

Survival in early- and late-term infants with congenital diaphragmatic hernia treated with extracorporeal membrane oxygenation.

BACKGROUND: Congenital diaphragmatic hernia (CDH) is a malformation of the diaphragm that allows bowel to enter the thoracic cavity, resulting in pulmonary hypoplasia and pulmonary hypertension. Approximately 50% of CDH patients are treated with extracorporeal membrane oxygenation (ECMO). The optimal gestational age for delivery of term infants with CDH at high risk for requiring ECMO is not known. The goal of this study was to compare survival of infants with CDH receiving ECMO born early term (38 0/7-39 6/7 weeks' gestation) with those born late term (40 0/7-41 6/7 weeks' gestation). Changes in survival rates of term infants and the factors associated with these changes were assessed over the 25 years that ECMO has been available. DESIGN: Retrospective cohort study of infants with CDH treated with ECMO. DATA SOURCES: The Extracorporeal Life Support Organization registry of patients treated at active Extracorporeal Life Support Organization centers from April 1976 through June 2001. ANALYSIS: Survival and clinical predictors of survival were compared between infants born early term (38 0/7-39 6/7 weeks' gestation) and infants born late term (40 0/7-41 6/7 weeks' gestation). Changes in survival rates over time and factors associated with survival were evaluated. RESULTS: Among full-term infants with CDH treated with ECMO, late-term compared with early-term delivery was associated with improved survival (63% vs 53%). Among full-term survivors of ECMO, late-term infants spent less time on ECMO (181 vs 197 hours) and less time in the hospital (60 vs 67 days). In multivariate analysis, greater birth weight, higher 5-minute Apgar score, higher arterial pH and PCO(2) <50 torr before ECMO, and absence of a prenatal diagnosis of CDH were associated with survival. Since the late 1980s, survival of infants with CDH requiring ECMO decreased from 63% to 52%. The decreased survival rate was associated with increased rates of prenatal diagnosis, early-term delivery, lower birth weight, longer ECMO runs, and more frequent complications on ECMO. CONCLUSIONS: Among term infants with CDH receiving ECMO, late-term delivery compared with early-term delivery is associated with improved survival, shorter ECMO duration, shorter hospital length of stay, and fewer complications on ECMO. These data suggest that, at least for the approximately 50% of CDH patients treated with ECMO, outcomes for infants with CDH may be improved by delay of elective delivery until 40 completed weeks of gestation.

The role of vascular growth factors in hyperoxia-induced injury to the developing lung.

Normal pulmonary vascular development is the result of a complex interplay of growth factors, including vascular endothelial growth factor (VEGF) and the angiopoietins. Injury to the developing lung, whether due to hyperoxia or mechanical ventilation, results in disordered vascular development, ranging from an apparent arrest of microvascular development in milder injury to extensive microvascular derangement in more severe injury. Alterations in vascular growth factors may participate in these injuries. During injury to the developing animal lung, VEGF abundance is markedly decreased. In models of post-injury recovery, up-regulation of VEGF accompanies the re-establishment of normal vasculature. Alterations in lung VEGF levels in human premature infants are less clear cut. However, among humans premature newborns who later go on to develop bronchopulmonary dysplasia (BPD), VEGF production is decreased in comparison to those newborns who recover. Other angiogenic factors, such as the CXC ELR+ chemokines, are also altered in injury to the developing lung, but their specific roles in vascular injury are less clear. Strategies that enhance microvascular integrity, whether through attenuating alterations in vascular growth factors or by other means, also improve the outcome of lung injury. Such therapies may eventually offer hope in human BPD.

Angiogenic factors and alveolar vasculature: development and alterations by injury in very premature baboons.

Proper formation of the pulmonary microvasculature is essential for normal lung development and gas exchange. Lung microvascular development may be disrupted by chronic injury of developing lungs in clinical diseases such as bronchopulmonary dysplasia. We examined microvascular development, angiogenic growth factors, and endothelial cell receptors in a fetal baboon model of chronic lung disease (CLD). In the last third of gestation, the endothelial cell marker platelet endothelial cell adhesion molecule (PECAM)-1 increased 7.5-fold, and capillaries immunostained for PECAM-1 changed from a central location in airspace septa to a subepithelial location. In premature animals delivered at 67% of term and supported with oxygen and ventilation for 14 days, PECAM-1 protein and capillary density did not increase, suggesting failure to expand the capillary network. The capillaries of the CLD animals were dysmorphic and not subepithelial. The angiogenic growth factor vascular endothelial growth factor (VEGF) and its receptor fms-like tyrosine kinase receptor (Flt-1) were significantly decreased in CLD. Angiopoietin-1, another angiogenic growth factor, and its receptor tyrosine kinase with immunoglobulin and epidermal growth factor homology domains were not significantly changed. These data suggest that CLD impairs lung microvascular development and that a possible mechanism is disruption of VEGF and Flt-1 expression.