Anne Hilgendorff

Prolonged mechanical ventilation with air induces apoptosis and causes failure of alveolar septation and angiogenesis in lungs of newborn mice

Defective lung septation and angiogenesis, quintessential features of neonatal chronic lung disease (CLD), typically result from lengthy exposure of developing lungs to mechanical ventilation (MV) and hyperoxia. Previous studies showed fewer alveoli and microvessels, with reduced VEGF and increased transforming growth factor-beta (TGFbeta) signaling, and excess, scattered elastin in lungs of premature infants and lambs with CLD vs. normal controls. MV of newborn mice with 40% O(2) for 24 h yielded similar lung structural abnormalities linked to impaired VEGF signaling, dysregulated elastin production, and increased apoptosis. These studies could not determine the relative importance of cyclic stretch vs. hyperoxia in causing these lung growth abnormalities. We therefore studied the impact of MV for 24 h with air on alveolar septation (quantitative lung histology), angiogenesis [CD31 quantitative-immunohistochemistry (IHC), immunoblots], apoptosis [TdT-mediated dUTP nick end labeling (TUNEL), active caspase-3 assays], VEGF signaling [VEGF-A, VEGF receptor 1 (VEGF-R1), VEGF-R2 immunoblots], TGFbeta activation [phosphorylated Smad2 (pSmad2) quantitative-IHC], and elastin production (tropoelastin immunoblots, quantitative image analysis of Harts stained sections) in lungs of 6-day-old mice. Compared with unventilated controls, MV caused a 3-fold increase in alveolar area, approximately 50% reduction in alveolar number and endothelial surface area, >5-fold increase in apoptosis, >50% decrease in lung VEGF-R2 protein, 4-fold increase of pSmad2 protein, and >50% increase in lung elastin, which was distributed throughout alveolar walls rather than at septal tips. This study is the first to show that prolonged MV of developing lungs, without associated hyperoxia, can inhibit alveolar septation and angiogenesis and increase apoptosis and lung elastin, findings that could reflect stretch-induced changes in VEGF and TGFbeta signaling, as reported in CLD.

Inhibiting Lung Elastase Activity Enables Lung Growth in Mechanically Ventilated Newborn Mice

RATIONALE Mechanical ventilation with O₂-rich gas (MV-O₂) offers life-saving treatment for respiratory failure, but also promotes lung injury. We previously reported that MV-O2 of newborn mice increased lung elastase activity, causing elastin degradation and redistribution of elastic fibers from septal tips to alveolar walls. These changes were associated with transforming growth factor (TGF)-β activation and increased apoptosis leading to defective alveolarization and lung growth arrest, as seen in neonatal chronic lung disease. OBJECTIVES To determine if intratracheal treatment of newborn mice with the serine elastase inhibitor elafin would prevent MV-O₂-induced lung elastin degradation and the ensuing cascade of events causing lung growth arrest. METHODS Five-day-old mice were treated via tracheotomy with recombinant human elafin or vehicle (lactated-Ringer solution), followed by MV with 40% O₂ for 8-24 hours; control animals breathed 40% O₂ without MV. At studys end, lungs were harvested to assess key variables noted below. MEASUREMENTS AND MAIN RESULTS MV-O₂ of vehicle-treated pups increased lung elastase and matrix metalloproteinase-9 activity when compared with unventilated control animals, causing elastin degradation (urine desmosine doubled), TGF-β activation (pSmad-2 tripled), and apoptosis (cleaved-caspase-3 increased 10-fold). Quantitative lung histology showed larger and fewer alveoli, greater inflammation, and scattered elastic fibers. Elafin blocked these MV-O₂-induced changes. CONCLUSIONS Intratracheal elafin, by blocking lung protease activity, prevented MV-O₂-induced elastin degradation, TGF-β activation, apoptosis, and dispersion of matrix elastin, and attenuated lung structural abnormalities noted in vehicle-treated mice after 24 hours of MV-O₂. These findings suggest that elastin breakdown contributes to defective lung growth in response to MV-O₂ and might be targeted therapeutically to prevent MV-O₂-induced lung injury.

Executive summary. Expert consensus statement on the diagnosis and treatment of paediatric pulmonary hypertension. The European Paediatric Pulmonary Vascular Disease Network, endorsed by ISHLT and DGPK

The European Paediatric Pulmonary Vascular Disease (PVD) Network is a registered, non-profit organisation that strives to define and develop effective, innovative diagnostic methods and treatment options in all forms of paediatric pulmonary hypertensive vascular disease, including specific forms such as pulmonary arterial hypertension (PAH)-congenital heart disease, pulmonary hypertension (PH) associated with bronchopulmonary dysplasia, persistent PH of the newborn, and related cardiac dysfunction. Methods The writing group members conducted searches of the PubMed/MEDLINE bibliographic database (1990–2015) and held five face-to-face meetings with votings. Clinical trials, guidelines, and reviews limited to paediatric data were searched using the terms ‘pulmonary hypertensioń’ and 5–10 other keywords, as outlined in the other nine articles of this special issue. Class of recommendation (COR) and level of evidence (LOE) were assigned based on European Society of Cardiology/American Heart Association definitions and on paediatric data only, or on adult studies that included >10% children. Results A total of 9 original consensus articles with graded recommendations (COR/LOE) were developed, and are summarised here. The topics included diagnosis/monitoring, genetics/biomarker, cardiac catheterisation, echocardiography, cardiac magnetic resonance/chest CT, associated forms of PH, intensive care unit/ventricular assist device/lung transplantation, and treatment of paediatric PAH. Conclusions The multipaper expert consensus statement of the European Paediatric PVD Network provides a specific, comprehensive, detailed but practical framework for the optimal clinical care of children with PH.

Neonatal mice genetically modified to express the elastase inhibitor elafin are protected against the adverse effects of mechanical ventilation on lung growth

Mechanical ventilation (MV) with O(2)-rich gas (MV-O(2)) offers life-saving treatment for newborn infants with respiratory failure, but it also can promote lung injury, which in neonates translates to defective alveolar formation and disordered lung elastin, a key determinant of lung growth and repair. Prior studies in preterm sheep and neonatal mice showed that MV-O(2) stimulated lung elastase activity, causing degradation and remodeling of matrix elastin. These changes yielded an inflammatory response, with TGF-β activation, scattered elastic fibers, and increased apoptosis, culminating in defective alveolar septation and arrested lung growth. To see whether sustained inhibition of elastase activity would prevent these adverse pulmonary effects of MV-O(2), we did studies comparing wild-type (WT) and mutant neonatal mice genetically modified to express in their vascular endothelium the human serine elastase inhibitor elafin (Eexp). Five-day-old WT and Eexp mice received MV with 40% O(2) (MV-O(2)) for 24-36 h. WT and Eexp controls breathed 40% O(2) without MV. MV-O(2) increased lung elastase and MMP-9 activity, resulting in elastin degradation (urine desmosine doubled), TGF-β activation (pSmad-2 increased 6-fold), apoptosis (cleaved-caspase-3 increased 10-fold), and inflammation (NF-κB activation, influx of neutrophils and monocytes) in lungs of WT vs. unventilated controls. These changes were blocked or blunted during MV-O(2) of Eexp mice. Scattered lung elastin and emphysematous alveoli observed in WT mice after 36 h of MV-O(2) were attenuated in Eexp mice. Both WT and Eexp mice showed defective VEGF signaling (decreased lung VEGF-R2 protein) and loss of pulmonary microvessels after lengthy MV-O(2), suggesting that elafins beneficial effects during MV-O(2) derived primarily from preserving matrix elastin and suppressing lung inflammation, thereby enabling alveolar formation during MV-O(2). These results suggest that degradation and remodeling of lung elastin can contribute to defective lung growth in response to MV-O(2) and might be targeted therapeutically to prevent ventilator-induced neonatal lung injury.

Bronchopulmonary dysplasia early changes leading to long-term consequences

Neonatal chronic lung disease, i.e., bronchopulmonary dysplasia, is characterized by impaired pulmonary development resulting from the impact of different risk factors including infections, hyperoxia, and mechanical ventilation on the immature lung. Remodeling of the extracellular matrix, apoptosis as well as altered growth factor signaling characterize the disease. The immediate consequences of these early insults have been studied in different animal models supported by results from in vitro approaches leading to the successful application of some findings to the clinical setting in the past. Nonetheless, existing information about long-term consequences of the identified early and most likely sustained changes to the developing lung is limited. Interesting results point towards a tremendous impact of these early injuries on the pulmonary repair capacity as well as aging related processes in the adult lung.

Pulmonary hypertension associated with acute or chronic lung diseases in the preterm and term neonate and infant. The European Paediatric Pulmonary Vascular Disease Network, endorsed by ISHLT and DGPK

Persistent pulmonary hypertension of the newborn (PPHN) is the most common neonatal form and mostly reversible after a few days with improvement of the underlying pulmonary condition. When pulmonary hypertension (PH) persists despite adequate treatment, the severity of parenchymal lung disease should be assessed by chest CT. Pulmonary vein stenosis may need to be ruled out by cardiac catheterisation and lung biopsy, and genetic workup is necessary when alveolar capillary dysplasia is suspected. In PPHN, optimisation of the cardiopulmonary situation including surfactant therapy should aim for preductal SpO2 between 91% and 95% and severe cases without post-tricuspid-unrestrictive shunt may receive prostaglandin E1 to maintain ductal patency in right heart failure. Inhaled nitric oxide is indicated in mechanically ventilated infants to reduce the need for extracorporal membrane oxygenation (ECMO), and sildenafil can be considered when this therapy is not available. ECMO may be indicated according to the ELSO guidelines. In older preterm infant, where PH is mainly associated with bronchopulmonary dysplasia (BPD) or in term infants with developmental lung anomalies such as congenital diaphragmatic hernia or cardiac anomalies, left ventricular diastolic dysfunction/left atrial hypertension or pulmonary vein stenosis, can add to the complexity of the disease. Here, oral or intravenous sildenafil should be considered for PH treatment in BPD, the latter for critically ill patients. Furthermore, prostanoids, mineralcorticoid receptor antagonists, and diuretics can be beneficial. Infants with proven or suspected PH should receive close follow-up, including preductal/postductal SpO2 measurements, echocardiography and laboratory work-up including NT-proBNP, guided by clinical improvement or lack thereof.

Bronchopulmonary dysplasia - an overview about pathophysiologic concepts

Neonatal chronic lung disease in the preterm infant, i.e. bronchopulmonary dysplasia (BPD) is characterized by impaired pulmonary development with its effects persisting into adulthood. Triggered in the immature lung by infectious complications, oxygen toxicity and the impact of mechanical ventilation, a sustained inflammatory response, extensive remodeling of the extracellular matrix, increased apoptosis as well as altered growth factor signaling characterize the disease. The current review focuses on selected pathophysiologic processes and their interplay in disease development. Furthermore, the potential of both, acute and long-term changes to the pulmonary scaffold and the cellular interface in concert with dysregulated growth factor signaling to affect aging and repair processes in the adult lung is discussed.

Visualization of neonatal lung injury associated with mechanical ventilation using x-ray dark-field radiography

Mechanical ventilation (MV) and supplementation of oxygen-enriched gas, often needed in postnatal resuscitation procedures, are known to be main risk factors for impaired pulmonary development in the preterm and term neonates. Unfortunately, current imaging modalities lack in sensitivity for the detection of early stage lung injury. The present study reports a new imaging approach for diagnosis and staging of early lung injury induced by MV and hyperoxia in neonatal mice. The imaging method is based on the Talbot-Lau x-ray grating interferometry that makes it possible to quantify the x-ray small-angle scattering on the air-tissue interfaces. This so-called dark-field signal revealed increasing loss of x-ray small-angle scattering when comparing images of neonatal mice undergoing hyperoxia and MV-O2 with animals kept at room air. The changes in the dark field correlated well with histologic findings and provided superior differentiation than conventional x-ray imaging and lung function testing. The results suggest that x-ray dark-field radiography is a sensitive tool for assessing structural changes in the developing lung. In the future, with further technical developments x-ray dark-field imaging could be an important tool for earlier diagnosis and sensitive monitoring of lung injury in neonates requiring postnatal oxygen or ventilator therapy.

Linking bronchopulmonary dysplasia to adult chronic lung diseases: role of WNT signaling.

Bronchopulmonary dysplasia (BPD) is one of the most common chronic lung diseases in infants caused by pre- and/or postnatal lung injury. BPD is characterized by arrested alveolarization and vascularization due to extracellular matrix remodeling, inflammation, and impaired growth factor signaling. WNT signaling is a critical pathway for normal lung development, and its altered signaling has been shown to be involved in the onset and progression of incurable chronic lung diseases in adulthood, such as chronic obstructive pulmonary disease (COPD) or idiopathic pulmonary fibrosis (IPF). In this review, we summarize the impact of WNT signaling on different stages of lung development and its potential contribution to developmental lung diseases, especially BPD, and chronic lung diseases in adulthood.

Genetic testing and blood biomarkers in paediatric pulmonary hypertension. Expert consensus statement on the diagnosis and treatment of paediatric pulmonary hypertension. The European Paediatric Pulmonary Vascular Disease Network, endorsed by ISHLT and DGPK

Childhood-onset pulmonary arterial hypertension (PAH) is considered complex and multifactorial, with relatively poor estimates of the natural history of the disease. Strategies allowing earlier detection, establishment of disease aetiology together with more accurate and sensitive biomarkers could enable better estimates of prognosis and individualise therapeutic strategies. Evidence is accumulating that genetic defects play an important role in the pathogenesis of idiopathic and hereditary forms of PAH. Altogether nine genes have been reported so far to be associated with childhood onset PAH suggesting that comprehensive multigene diagnostics can be useful in the assessment. Identification of disease-causing mutations allows estimates of prognosis and forms the most effective way for risk stratification in the family. In addition to genetic determinants the analysis of blood biomarkers are increasingly used in clinical practice to evaluate disease severity and treatment responses. As in genetic diagnostics, a multiplex approach can be helpful, as a single biomarker for PAH is unlikely to meet all requirements. This consensus statement reviews the current evidence for the use of genetic diagnostics and use of blood biomarkers in the assessment of paediatric patients with PAH.