Neonatal Lung Disease Associated with TBX4 Mutations.

Variable lung disease was documented in 2 infants with heterozygous TBX4 mutations; their clinical presentations, pathology, and outcomes were distinct. These findings demonstrate that TBX4 gene mutations are associated with neonatal respiratory failure and highlight the wide spectrum of clinicopathological outcomes that have implications for patient diagnosis and management.

Neonatal hyperoxia promotes asthma-like features through IL-33-dependent ILC2 responses.

BACKGROUND: Premature infants often require oxygen supplementation and, therefore, are exposed to oxidative stress. Following oxygen exposure, preterm infants frequently develop chronic lung disease and have a significantly increased risk of asthma. OBJECTIVE: We sought to identify the underlying mechanisms by which neonatal hyperoxia promotes asthma development. METHODS: Mice were exposed to neonatal hyperoxia followed by a period of room air recovery. A group of mice was also intranasally exposed to house dust mite antigen. Assessments were performed at various time points for evaluation of airway hyperresponsiveness, eosinophilia, mucus production, inflammatory gene expression, and TH and group 2 innate lymphoid cell (ILC2) responses. Sera from term- and preterm-born infants were also collected and levels of IL-33 and type 2 cytokines were measured. RESULTS: Neonatal hyperoxia induced asthma-like features including airway hyperresponsiveness, mucus hyperplasia, airway eosinophilia, and type 2 pulmonary inflammation. In addition, neonatal hyperoxia promoted allergic TH responses to house dust mite exposure. Elevated IL-33 levels and ILC2 responses were observed in the lungs most likely due to oxidative stress caused by neonatal hyperoxia. IL-33 receptor signaling and ILC2s were vital for the induction of asthma-like features following neonatal hyperoxia. Serum IL-33 levels correlated significantly with serum levels of IL-5 and IL-13 but not IL-4 in preterm infants. CONCLUSIONS: These data demonstrate that an axis involving IL-33 and ILC2s is important for the development of asthma-like features following neonatal hyperoxia and suggest therapeutic potential for targeting IL-33, ILC2s, and oxidative stress to prevent and/or treat asthma development related to prematurity.

Initial Suppression of Transforming Growth Factor-ß Signaling and Loss of TGFBI Causes Early Alveolar Structural Defects Resulting in Bronchopulmonary Dysplasia.

Septation of the gas-exchange saccules of the morphologically immature mouse lung requires regulated timing, spatial direction, and dosage of transforming growth factor (TGF)-ß signaling. We found that neonatal hyperoxia acutely initially diminished saccular TGF-ß signaling coincident with alveolar simplification. However, sustained hyperoxia resulted in a biphasic response and subsequent up-regulation of TGF-ß signaling, ultimately resulting in bronchopulmonary dysplasia. Significantly, we found that the TGF-ß-induced matricellular protein (TGFBI) was similarly biphasically altered in response to hyperoxia. Moreover, genetic ablation revealed that TGFBI was required for normal alveolar structure and function. Although the phenotype was not neonatal lethal, Tgfbi-deficient lungs were morphologically abnormal. Mutant septal tips were stunted, lacked elastin-positive tips, exhibited reduced proliferation, and contained abnormally persistent alveolar α-smooth muscle actin myofibroblasts. In addition, Tgfbi-deficient lungs misexpressed TGF-ß-responsive follistatin and serpine 1, and transiently suppressed myofibroblast platelet-derived growth factor α differentiation marker. Finally, despite normal lung volume, Tgfbi-null lungs displayed diminished elastic recoil and gas exchange efficiency. Combined, these data demonstrate that initial suppression of the TGF-ß signaling apparatus, as well as loss of key TGF-ß effectors (like TGFBI), underlies early alveolar structural defects, as well as long-lasting functional deficits routinely observed in chronic lung disease of infancy patients. These studies underline the complex (and often contradictory) role of TGF-ß and indicate a need to design studies to associate alterations with initial appearance of phenotypical changes suggestive of bronchopulmonary dysplasia.

Relationship of structural to functional impairment during alveolar-capillary membrane development.

Bronchopulmonary dysplasia is a chronic lung disease of extreme preterm infants and results in impaired gas exchange. Although bronchopulmonary dysplasia is characterized histologically by alveolar-capillary simplification in animal models, it is clinically defined by impaired gas diffusion. With the use of a developmentally relevant model, we correlated alveolar-capillary structural simplification with reduced functional gas exchange as measured by the diffusing factor for carbon monoxide (DFCO). Neonatal mouse pups were exposed to >90% hyperoxia or room air during postnatal days 0 to 7, and then all pups were returned to room air from days 7 to 56. At day 56, DFCO was measured as the ratio of carbon monoxide uptake to neon dilution, and lungs were fixed for histologic assessment of alveolar-capillary development. Neonatal hyperoxia exposure inhibited alveolar-capillary septal development as evidenced by significantly increased mean linear intercept, increased airspace-to-septal ratio, decreased nodal density, and decreased pulmonary microvasculature. Importantly, alveolar-capillary structural deficits in hyperoxia-exposed pups were accompanied by a significant 28% decrease in DFCO (0.555 versus 0.400; P < 0.0001). In addition, DFCO was highly and significantly correlated with structural measures of reduced alveolar-capillary growth. Simplification of alveolar-capillary structure is highly correlated with impaired gas exchange function. Current mechanistic and therapeutic animal models of inhibited alveolar development may benefit from application of DFCO as an alternative physiologic indicator of alveolar-capillary development.

Neonatal hyperoxic lung injury favorably alters adult right ventricular remodeling response to chronic hypoxia exposure.

The development of pulmonary hypertension (PH) requires multiple pulmonary vascular insults, yet the role of early oxygen therapy as an initial pulmonary vascular insult remains poorly defined. Here, we employ a two-hit model of PH, utilizing postnatal hyperoxia followed by adult hypoxia exposure, to evaluate the role of early hyperoxic lung injury in the development of later PH. Sprague-Dawley pups were exposed to 90% oxygen during postnatal days 0-4 or 0-10 or to room air. All pups were then allowed to mature in room air. At 10 wk of age, a subset of rats from each group was exposed to 2 wk of hypoxia (Patm = 362 mmHg). Physiological, structural, and biochemical endpoints were assessed at 12 wk. Prolonged (10 days) postnatal hyperoxia was independently associated with elevated right ventricular (RV) systolic pressure, which worsened after hypoxia exposure later in life. These findings were only partially explained by decreases in lung microvascular density. Surprisingly, postnatal hyperoxia resulted in robust RV hypertrophy and more preserved RV function and exercise capacity following adult hypoxia compared with nonhyperoxic rats. Biochemically, RVs from animals exposed to postnatal hyperoxia and adult hypoxia demonstrated increased capillarization and a switch to a fetal gene pattern, suggesting an RV more adept to handle adult hypoxia following postnatal hyperoxia exposure. We concluded that, despite negative impacts on pulmonary artery pressures, postnatal hyperoxia exposure may render a more adaptive RV phenotype to tolerate late pulmonary vascular insults.

Assessment of inhibited alveolar-capillary membrane structural development and function in bronchopulmonary dysplasia.

Bronchopulmonary dysplasia (BPD) is a chronic lung disease of extreme prematurity and is defined clinically by dependence on supplemental oxygen due to impaired gas exchange. Optimal gas exchange is dependent on the development of a sufficient surface area for diffusion. In the mammalian lung, rapid acquisition of distal lung surface area is accomplished in neonatal and early adult life by means of vascularization and secondary septation of distal lung airspaces. Extreme preterm birth interrupts secondary septation and pulmonary capillary development and ultimately reduces the efficiency of the alveolar-capillary membrane. Although pulmonary health in BPD infants rapidly improves over the first few years, persistent alveolar-capillary membrane dysfunction continues into adolescence and adulthood. Preventative therapies have been largely ineffective, and therapies aimed at promoting normal development of the air-blood barrier in infants with established BPD remain largely unexplored. The purpose of this review will be: (1) to summarize the histological evidence of aberrant alveolar-capillary membrane development associated with extreme preterm birth and BPD, (2) to review the clinical evidence assessing the long-term impact of BPD on alveolar-capillary membrane function, and (3) to discuss the need to develop and incorporate direct measurements of functional gas exchange into clinically relevant animal models of inhibited alveolar development.

Periostin downregulation is an early marker of inhibited neonatal murine lung alveolar septation.

BACKGROUND: Extreme preterm birth exposes the saccular lung to multiple teratogens, which ultimately retard alveolar development. Specifically, therapeutic high level oxygen supplementation adversely affects the premature lungs and results in blunted alveolarization. Prolonged hyperoxic lung injury has previously been shown to upregulate the matricellular protein Periostin (Postn) and stimulate ectopic accumulation of alpha smooth muscle actin (αSMA) myofibroblasts. Therapies that promote lung septation are lacking largely due to a lack of reliable early biomarkers of injury. Thus, we determined if Postn expression correlated with the initial appearance of myofibroblasts in the saccular lung and was required for early alveolar development. METHODS: Lung development in C57BL/6J mice following room-air (RA, 21%-O2) or continuous hyperoxia (85%-O2) from birth (P0) through postnatal day P14 was correlated with Postn and αSMA expression. Alveolarization in Postn knockout mice exposed to room-air, 60%-, and 85%-O2 was also examined. RESULTS: Postn was widely expressed in distal lung septa through P2 to P4 and peak expression coincided with accumulation of saccular myofibroblasts. Initially, 85%-O2 prematurely downregulated Postn and αSMA expression and suppressed proliferation before the first evidence of distal lung simplification at P4. By P14, chronic 85%-O2 resulted in secondary upregulation of Postn and αSMA in blunted septa. Myofibroblast differentiation and alveolar development was unaffected in Postn null mice and acute 85%-O2 exposure equally inhibited septal formation in Postn null and wild-type littermates. CONCLUSION: Postn expression is tightly correlated with the presence of αSMA-myofibroblasts and is a novel early biomarker of acutely inhibited alveolar septation during a crucial window of lung development.

Periostin regulates goblet cell metaplasia in a model of allergic airway inflammation.

Periostin is a 90-kDa member of the fasciclin-containing family and functions as part of the extracellular matrix. Periostin is expressed in a variety of tissues and expression is increased in airway epithelial cells from asthmatic patients. Recent studies have implicated a role for periostin in allergic eosinophilic esophagitis. To further define a role for periostin in Th2-mediated inflammatory diseases such as asthma, we studied the development of allergic pulmonary inflammation in periostin-deficient mice. Sensitization and challenge of periostin-deficient mice with OVA resulted in increased peripheral Th2 responses compared with control mice. In the lungs, periostin deficiency resulted in increased airway resistance and significantly enhanced mucus production by goblet cells concomitant with increased expression of Gob5 and Muc5ac compared with wild type littermates. Periostin also inhibited the expression of Gob5, a putative calcium-activated chloride channel involved in the regulation of mucus production, in primary murine airway epithelial cells. Our studies suggest that periostin may be part of a negative-feedback loop regulating allergic inflammation that could be therapeutic in the treatment of atopic disease.

Respiratory support strategies in the prevention and treatment of bronchopulmonary dysplasia.

Neonates who are born preterm frequently have inadequate lung development to support independent breathing and will need respiratory support. The underdeveloped lung is also particularly susceptible to lung injury, especially during the first weeks of life. Consequently, respiratory support strategies in the early stages of premature lung disease focus on minimizing alveolar damage. As infants grow and lung disease progresses, it becomes necessary to shift respiratory support to a strategy targeting the often severe pulmonary heterogeneity and obstructive respiratory physiology. With appropriate management, time, and growth, even those children with the most extreme prematurity and severe lung disease can be expected to wean from respiratory support.

Periostin, a matricellular protein, plays a role in the induction of chemokines in pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and usually fatal form of interstitial lung disease (ILD). The precise molecular mechanisms of IPF remain poorly understood. However, analyses of mice receiving bleomycin (BLM) as a model of IPF established the importance of preceding inflammation for the formation of fibrosis. Periostin is a recently characterized matricellular protein involved in modulating cell functions. We recently found that periostin is highly expressed in the lung tissue of patients with IPF, suggesting that it may play a role in the process of pulmonary fibrosis. To explore this possibility, we administered BLM to periostin-deficient mice, and they subsequently showed a reduction of pulmonary fibrosis. We next determined whether this result was caused by a decrease in the preceding recruitment of neutrophils and macrophages in the lungs because of the lower production of chemokines and proinflammatory cytokines. We performed an in vitro analysis of chemokine production in lung fibroblasts, which indicated that periostin-deficient fibroblasts produced few or no chemokines in response to TNF-α compared with control samples, at least partly explaining the lack of inflammatory response and, therefore, fibrosis after BLM administration to periostin-deficient mice. In addition, we confirmed that periostin is highly expressed in the lung tissue of chemotherapeutic-agent-induced ILD as well as of patients with IPF. Taking these results together, we conclude that periostin plays a unique role as an inducer of chemokines to recruit neutrophils and macrophages important in the process of pulmonary fibrosis in BLM-administered model mice. Our results suggest a therapeutic potential for periostin in IPF and drug-induced ILD.

Aberrant signaling pathways of the lung mesenchyme and their contributions to the pathogenesis of bronchopulmonary dysplasia.

Bronchopulmonary dysplasia (BPD) is a chronic lung disease in infants born extremely preterm, typically before 28 weeks' gestation, characterized by a prolonged need for supplemental oxygen or positive pressure ventilation beyond 36 weeks postmenstrual age. The limited number of autopsy samples available from infants with BPD in the postsurfactant era has revealed a reduced capacity for gas exchange resulting from simplification of the distal lung structure with fewer, larger alveoli because of a failure of normal lung alveolar septation and pulmonary microvascular development. The mechanisms responsible for alveolar simplification in BPD have not been fully elucidated, but mounting evidence suggests that aberrations in the cross-talk between growth factors of the lung mesenchyme and distal airspace epithelium have a key role. Animal models that recapitulate the human condition have expanded our knowledge of the pathology of BPD and have identified candidate matrix components and growth factors in the developing lung that are disrupted by conditions that predispose infants to BPD and interfere with normal vascular and alveolar morphogenesis. This review focuses on the deviations from normal lung development that define the pathophysiology of BPD and summarizes the various candidate mesenchyme-associated proteins and growth factors that have been identified as being disrupted in animal models of BPD. Finally, future areas of research to identify novel targets affected in arrested lung development and recovery are discussed.

Maladaptive functional changes in alveolar fibroblasts due to perinatal hyperoxia impair epithelial differentiation.

Infants born prematurely worldwide have up to a 50% chance of developing bronchopulmonary dysplasia (BPD), a clinical morbidity characterized by dysregulated lung alveolarization and microvascular development. It is known that PDGFR alpha-positive (PDGFRA+) fibroblasts are critical for alveolarization and that PDGFRA+ fibroblasts are reduced in BPD. A better understanding of fibroblast heterogeneity and functional activation status during pathogenesis is required to develop mesenchymal population-targeted therapies for BPD. In this study, we utilized a neonatal hyperoxia mouse model (90% O2 postnatal days 0-7, PN0-PN7) and performed studies on sorted PDGFRA+ cells during injury and room air recovery. After hyperoxia injury, PDGFRA+ matrix and myofibroblasts decreased and PDGFRA+ lipofibroblasts increased by transcriptional signature and population size. PDGFRA+ matrix and myofibroblasts recovered during repair (PN10). After 7 days of in vivo hyperoxia, PDGFRA+ sorted fibroblasts had reduced contractility in vitro, reflecting loss of myofibroblast commitment. Organoids made with PN7 PDGFRA+ fibroblasts from hyperoxia in mice exhibited reduced alveolar type 1 cell differentiation, suggesting reduced alveolar niche-supporting PDGFRA+ matrix fibroblast function. Pathway analysis predicted reduced WNT signaling in hyperoxia fibroblasts. In alveolar organoids from hyperoxia-exposed fibroblasts, WNT activation by CHIR increased the size and number of alveolar organoids and enhanced alveolar type 2 cell differentiation.

Tracheostomy prediction model in neonatal bronchopulmonary dysplasia via lung and airway MRI.

RATIONALE: Clinical management of neonatal bronchopulmonary dysplasia (BPD) is often imprecise and can vary widely between different institutions and providers, due to limited objective measurements of disease pathology severity. There is critical need to improve guidance on the application and timing of interventional treatments, such as tracheostomy. OBJECTIVES: To generate an imaging-based clinical tool for early identification of those patients with BPD who are likely to require later tracheostomy and long-term mechanical ventilation. METHODS: We conducted a prospective cohort study of n = 61 infants (55 BPD, 6 preterm non-BPD). Magnetic resonance imaging (MRI) scores of lung parenchymal disease were used to create a binomial logistic regression model for predicting tracheostomy requirement. This model was further investigated using clinical variables and MRI-quantified tracheomalacia (TM). MEASUREMENTS AND MAIN RESULTS: A model for predicting tracheostomy requirement was created using MRI parenchymal score. This model had 89% accuracy, 100% positive predictive value (PPV), and 85% negative predictive value (NPV), compared with 84%, 60%, and 83%, respectively, when using only relevant clinical variables. In a subset of patients with airway MRI (n = 36), a model including lung and TM measurements had 83% accuracy, 92% PPV, and 78% NPV. CONCLUSIONS: MRI-based measurements of parenchymal disease and TM can be used to predict need for tracheostomy in infants with BPD, more accurately than clinical factors alone. This prediction model has strong potential as a clinical tool for physicians and families for early determination of tracheostomy requirement.

Safety of sildenafil in extremely premature infants: a phase I trial.

OBJECTIVE: To characterize the safety of sildenafil in premature infants. STUDY DESIGN: A phase I, open-label trial of sildenafil in premature infants receiving sildenafil per usual clinical care (cohort 1) or receiving a single IV dose of sildenafil (cohort 2). Safety was evaluated based on adverse events (AEs), transaminase levels, and mean arterial pressure monitoring. RESULTS: Twenty-four infants in cohort 1 (n = 25) received enteral sildenafil. In cohort 2, infants received a single IV sildenafil dose of 0.25 mg/kg (n = 7) or 0.125 mg/kg (n = 2). In cohort 2, there was one serious AE related to study drug involving hypotension associated with a faster infusion rate than specified by the protocol. There were no AEs related to elevated transaminases. CONCLUSION: Sildenafil was well tolerated by the study population. Drug administration times and flush rates require careful attention to prevent infusion-related hypotension associated with faster infusions of IV sildenafil in premature infants. CLINICAL TRIAL: ClinicalTrials.gov Identifier: NCT01670136.

Morbidity and respiratory outcomes in infants requiring tracheostomy for severe bronchopulmonary dysplasia.

OBJECTIVE: The decision for tracheostomy for bronchopulmonary dysplasia (BPD) is highly variable and often dictated by local practice. We aimed to characterize morbidity, mortality, and respiratory outcomes in preterm infants undergoing tracheostomy for severe BPD. STUDY DESIGN: We retrospectively reviewed a single-center 4-year cohort of all infants born <33 weeks gestational age (GA) that required tracheostomy due to severe BPD. Indications for tracheostomy apart from BPD were excluded. Demographic information, comorbidities, respiratory management, age at tracheostomy, post-discharge respiratory outcomes, and survival were examined up to at least 5 years of age. RESULTS: At a mean corrected GA of 43.3 weeks, 49 preterm infants with severe BPD required tracheostomy. Forty-six infants (94%) had long-term follow-up. Compared to survivors, the 12 (26.1%) infants that died were significantly more likely to be small for gestational age (SGA) or require treatment for pulmonary hypertension. GA, birth weight, sex, antenatal corticosteroid exposure, need for patent ductus arteriosus ligation, and magnitude of respiratory support at tracheostomy placement were not associated with mortality. At the latest follow-up, 97% were liberated from mechanical ventilation and 79% decannulated. Morbidities of the upper airway were common, and 13/27 (47%) decannulated infants had required airway reconstruction. CONCLUSION: Preterm infants undergoing tracheostomy experienced significant mortality, particularly those who were SGA or had pulmonary hypertension. However, by 5 years of age, most infants liberalized from mechanical ventilation and decannulated. Magnitude of respiratory support at time of tracheostomy was not associated with mortality and should not deter intervention. Nearly half of patients required airway reconstruction before decannulation.

Cumulative effects of neonatal hyperoxia on murine alveolar structure and function.

BACKGROUND: Bronchopulmonary dysplasia (BPD) results from alveolar simplification and abnormal development of alveolar and capillary structure. Survivors of BPD display persistent deficits in airflow and membrane and vascular components of alveolar gas diffusion. Despite being the defining feature of BPD, various neonatal hyperoxia models of BPD have not routinely assessed pulmonary gas diffusion. METHODS: To simulate the most commonly-utilized neonatal hyperoxia models, we exposed neonatal mice to room air or ≥90% hyperoxia during key stages of distal lung development: through the first 4 (saccular), 7 (early alveolar), or 14 (bulk alveolar) postnatal days, followed by a period of recovery in room air until 8 weeks of age when alveolar septation is essentially complete. We systematically assessed and correlated the effects of neonatal hyperoxia on the degree of alveolar-capillary structural and functional impairment. We hypothesized that the degree of alveolar-capillary simplification would correlate strongly with worsening diffusion impairment. RESULTS: Neonatal hyperoxia exposure, of any duration, resulted in alveolar simplification and impaired pulmonary gas diffusion. Mean Linear Intercept increased in proportion to the length of hyperoxia exposure while alveolar and total lung volume increased markedly only with prolonged exposure. Surprisingly, despite having a similar effect on alveolar surface area, only prolonged hyperoxia for 14 days resulted in reduced pulmonary microvascular volume. Estimates of alveolar and capillary structure, in general, correlated poorly with assessment of gas diffusion. CONCLUSION: Our results help define the physiological and structural consequences of commonly-employed neonatal hyperoxia models of BPD and inform their clinical utility. Pediatr Pulmonol. 2017;52:616-624. © 2016 Wiley Periodicals, Inc.

Lung parenchymal development in premature infants without bronchopulmonary dysplasia.

RATIONALE: While infants who are born extremely premature and develop bronchopulmonary dysplasia (BPD) have impaired alveolar development and decreased pulmonary diffusion (DLCO), it remains unclear whether infants born less premature and do not develop BPD, healthy premature (HP), have impaired parenchymal development. In addition, there is increasing evidence that pro-angiogenic cells are important for vascular development; however, there is little information on the relationship of pro-angiogenic cells to lung growth and development in infants. OBJECTIVE: and Methods Determine among healthy premature (HP) and fullterm (FT) infants, whether DLCO and alveolar volume (VA) are related to gestational age at birth (GA), respiratory support during the neonatal period (mechanical ventilation [MV], supplemental oxygen [O2], continuous positive airway pressure [CPAP]), and pro-angiogenic circulating hematopoietic stem/progenitor cells (CHSPCs). We measured DLCO, VA, and CHSPCs in infants between 3-33 months corrected-ages; HP (mean GA = 31.7 wks; N = 48,) and FT (mean GA = 39.3 wks; N =88). RESULT: DLCO was significantly higher in HP than FT subjects, while there was no difference in VA , after adjusting for body length, gender, and race. DLCO and VA were not associated with GA, MV and O2; however, higher values were associated with higher CHSPCs, as well as treatment with CPAP. CONCLUSION: Our findings suggest that in the absence of extreme premature birth, as well as BPD, prematurity per se, does not impair lung parenchymal development.