Relationship between impaired BMP signalling and clinical risk factors at early-stage vascular injury in the preterm infant.

INTRODUCTION: Chronic lung disease, that is, bronchopulmonary dysplasia (BPD) is the most common complication in preterm infants and develops as a consequence of the misguided formation of the gas-exchange area undergoing prenatal and postnatal injury. Subsequent vascular disease and its progression into pulmonary arterial hypertension critically determines long-term outcome in the BPD infant but lacks identification of early, disease-defining changes. METHODS: We link impaired bone morphogenetic protein (BMP) signalling to the earliest onset of vascular pathology in the human preterm lung and delineate the specific effects of the most prevalent prenatal and postnatal clinical risk factors for lung injury mimicking clinically relevant conditions in a multilayered animal model using wild-type and transgenic neonatal mice. RESULTS: We demonstrate (1) the significant reduction in BMP receptor 2 (BMPR2) expression at the onset of vascular pathology in the lung of preterm infants, later mirrored by reduced plasma BMP protein levels in infants with developing BPD, (2) the rapid impairment (and persistent change) of BMPR2 signalling on postnatal exposure to hyperoxia and mechanical ventilation, aggravated by prenatal cigarette smoke in a preclinical mouse model and (3) a link to defective alveolar septation and matrix remodelling through platelet derived growth factor-receptor alpha deficiency. In a treatment approach, we partially reversed vascular pathology by BMPR2-targeted treatment with FK506 in vitro and in vivo. CONCLUSION: We identified impaired BMP signalling as a hallmark of early vascular disease in the injured neonatal lung while outlining its promising potential as a future biomarker or therapeutic target in this growing, high-risk patient population.

Attenuated PDGF signaling drives alveolar and microvascular defects in neonatal chronic lung disease.

Neonatal chronic lung disease (nCLD) affects a significant number of neonates receiving mechanical ventilation with oxygen-rich gas (MV-O2). Regardless, the primary molecular driver of the disease remains elusive. We discover significant enrichment for SNPs in the PDGF-Rα gene in preterms with nCLD and directly test the effect of PDGF-Rα haploinsufficiency on the development of nCLD using a preclinical mouse model of MV-O2 In the context of MV-O2, attenuated PDGF signaling independently contributes to defective septation and endothelial cell apoptosis stemming from a PDGF-Rα-dependent reduction in lung VEGF-A. TGF-ß contributes to the PDGF-Rα-dependent decrease in myofibroblast function. Remarkably, endotracheal treatment with exogenous PDGF-A rescues both the lung defects in haploinsufficient mice undergoing MV-O2 Overall, our results establish attenuated PDGF signaling as an important driver of nCLD pathology with provision of PDGF-A as a protective strategy for newborns undergoing MV-O2.

Facilitated Diagnosis of Pneumothoraces in Newborn Mice Using X-ray Dark-Field Radiography.

OBJECTIVE: The aim of this study was to evaluate the diagnostic value of x-ray dark-field imaging in projection radiography-based depiction of pneumothoraces in the neonatal murine lung, a potentially life-threatening medical condition that requires a timely and correct diagnosis. MATERIALS AND METHODS: By the use of a unique preclinical model, 7-day-old C57Bl/6N mice received mechanical ventilation for 2 or 8 hours with oxygen-rich gas (FIO2 = 0.4; n = 24). Unventilated mice either spontaneously breathed oxygen-rich gas (FIO2 = 0.4) for 2 or 8 hours or room air (n = 22). At the end of the experiment, lungs were inflated with a standardized volume of air after a lethal dose of pentobarbital was administered to the pups. All lungs were imaged with a prototype grating-based small-animal scanner to acquire x-ray transmission and dark-field radiographs. Image contrast between the air-filled pleural space and lung tissue was quantified for both transmission and dark-field radiograms. After the independent expert's assessment, 2 blinded readers evaluated all dark-field and transmission images for the presence or absence of pneumothoraces. Contrast ratios, diagnostic accuracy, as well as reader's confidence and interreader agreement were recorded for both imaging modalities. RESULTS: Evaluation of both x-ray transmission and dark-field radiographs by independent experts revealed the development of a total of 10 pneumothoraces in 8 mice. Here, the contrast ratio between the air-filled pleural space of the pneumothoraces and the lung tissue was significantly higher in the dark field (8.4 ± 3.5) when compared with the transmission images (5.1 ± 2.8; P < 0.05). Accordingly, the readers' diagnostic confidence for the diagnosis of pneumothoraces was significantly higher for dark-field compared with transmission images (P = 0.001). Interreader agreement improved from moderate for the analysis of transmission images alone (κ = 0.41) to very good when analyzing dark-field images alone (κ = 0.90) or in combination with transmission images (κ = 0.88). Diagnostic accuracy significantly improved for the analysis of dark-field images alone (P = 0.04) or in combination with transmission images (P = 0.02), compared with the analysis of transmission radiographs only. CONCLUSIONS: The significant improvement in contrast ratios between lung parenchyma and free air in the dark-field images allows the facilitated detection of pneumothoraces in the newborn mouse. These preclinical experiments indicate the potential of the technique for future clinical applications.

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.

Absence of TNF-α enhances inflammatory response in the newborn lung undergoing mechanical ventilation.

Bronchopulmonary dysplasia (BPD), characterized by impaired alveolarization and vascularization in association with lung inflammation and apoptosis, often occurs after mechanical ventilation with oxygen-rich gas (MV-O2). As heightened expression of the proinflammatory cytokine TNF-α has been described in infants with BPD, we hypothesized that absence of TNF-α would reduce pulmonary inflammation, and attenuate structural changes in newborn mice undergoing MV-O2 Neonatal TNF-α null (TNF-α(-/-)) and wild type (TNF-α(+/+)) mice received MV-O2 for 8 h; controls spontaneously breathed 40% O2 Histologic, mRNA, and protein analysis in vivo were complemented by in vitro studies subjecting primary pulmonary myofibroblasts to mechanical stretch. Finally, TNF-α level in tracheal aspirates from preterm infants were determined by ELISA. Although MV-O2 induced larger and fewer alveoli in both, TNF-α(-/-) and TNF-α(+/+) mice, it caused enhanced lung apoptosis (TUNEL, caspase-3/-6/-8), infiltration of macrophages and neutrophils, and proinflammatory mediator expression (IL-1ß, CXCL-1, MCP-1) in TNF-α(-/-) mice. These differences were associated with increased pulmonary transforming growth factor-ß (TGF-ß) signaling, decreased TGF-ß inhibitor SMAD-7 expression, and reduced pulmonary NF-κB activity in ventilated TNF-α(-/-) mice. Preterm infants who went on to develop BPD showed significantly lower TNF-α levels at birth. Our results suggest a critical balance between TNF-α and TGF-ß signaling in the developing lung, and underscore the critical importance of these key pathways in the pathogenesis of BPD. Future treatment strategies need to weigh the potential benefits of inhibiting pathologic cytokine expression against the potential of altering key developmental pathways.