To assess the utility of clinical predictors of persistent respiratory morbidity in extremely low gestational age newborns (ELGANs).
We enrolled ELGANs (<29 weeks' gestation) at ≤7 postnatal days and ...collected antenatal and neonatal clinical data through 36 weeks' postmenstrual age. We surveyed caregivers at 3, 6, 9, and 12 months' corrected age to identify postdischarge respiratory morbidity, defined as hospitalization, home support (oxygen, tracheostomy, ventilation), medications, or symptoms (cough/wheeze). Infants were classified as having postprematurity respiratory disease (PRD, the primary study outcome) if respiratory morbidity persisted over ≥2 questionnaires. Infants were classified with severe respiratory morbidity if there were multiple hospitalizations, exposure to systemic steroids or pulmonary vasodilators, home oxygen after 3 months or mechanical ventilation, or symptoms despite inhaled corticosteroids. Mixed-effects models generated with data available at 1 day (perinatal) and 36 weeks' postmenstrual age were assessed for predictive accuracy.
Of 724 infants (918 ± 234 g, 26.7 ± 1.4 weeks' gestational age) classified for the primary outcome, 68.6% had PRD; 245 of 704 (34.8%) were classified as severe. Male sex, intrauterine growth restriction, maternal smoking, race/ethnicity, intubation at birth, and public insurance were retained in perinatal and 36-week models for both PRD and respiratory morbidity severity. The perinatal model accurately predicted PRD (c-statistic 0.858). Neither the 36-week model nor the addition of bronchopulmonary dysplasia to the perinatal model improved accuracy (0.856, 0.860); c-statistic for BPD alone was 0.907.
Both bronchopulmonary dysplasia and perinatal clinical data accurately identify ELGANs at risk for persistent and severe respiratory morbidity at 1 year.
ClinicalTrials.gov: NCT01435187.
Alveolar type I (TI) cells are large squamous cells that cover >95% of the internal surface area of the lung; type II (TII) cells are small cuboidal cells with distinctive intracellular surfactant ...storage organelles. Based on autoradiographic studies in the 1970s, the long-held paradigm of alveolar epithelial development has been a linear progression from undifferentiated progenitor cells through TII cells to TI cells. Subsequent data support the existence of more complex pathways. Recently, a bipotent TI/TII progenitor cell at embryonic day E18 has been inferred both from marker expression in developing airways and from statistical analyses of gene expression data obtained from single-lung embryonic cells. To study cell lineage directly by fate mapping, we developed new transgenic mouse models in which rtTA is driven either by the rat podoplanin or the mouse Sftpc gene to mark cells irreversibly in development. Using these models, we found two distinct lineage pathways. One pathway, evident as early as E12-15, is devoted almost exclusively to TI cell development; a second pathway gives rise predominantly to TII cells but also a subpopulation of TI cells. We have defined the molecular phenotypes of these distinct progenitor populations and have identified potential regulatory factors in TI and TII cell differentiation. By analyzing gene pathways in mature TI and TII cells, we identified potential novel functions of each cell type. These results provide novel insights into lung development and suggest a basis for testing strategies to promote alveolar differentiation and repair, including potential transplantation of lineage-specific progenitor cells.
Claudins are a family of transmembrane proteins that are required for tight junction formation. Claudin (CLDN)-18.1, the only known lung-specific tight junction protein, is the most abundant claudin ...in alveolar epithelial type (AT) 1 cells, and is regulated by lung maturational agonists and inflammatory mediators. To determine the function of CLDN18 in the alveolar epithelium, CLDN18 knockout (KO) mice were generated and studied by histological, biochemical, and physiological approaches, in addition to whole-genome microarray. Alveolar epithelial barrier function was assessed after knockdown of CLDN18 in isolated lung cells. CLDN18 levels were measured by quantitative PCR in lung samples from fetal and postnatal human infants. We found that CLDN18 deficiency impaired alveolar epithelial barrier function in vivo and in vitro, with evidence of increased paracellular permeability and architectural distortion at AT1-AT1 cell junctions. Although CLDN18 KO mice were born without evidence of a lung abnormality, histological and gene expression analysis at Postnatal Day 3 and Week 4 identified impaired alveolarization. CLDN18 KO mice also had evidence of postnatal lung injury, including acquired AT1 cell damage. Human fetal lungs at 23-24 weeks gestational age, the highest-risk period for developing bronchopulmonary dysplasia, a disease of impaired alveolarization, had significantly lower CLDN18 expression relative to postnatal lungs. Thus, CLDN18 deficiency results in epithelial barrier dysfunction, injury, and impaired alveolarization in mice. Low expression of CLDN18 in human fetal lungs supports further investigation into a role for this tight junction protein in bronchopulmonary dysplasia.
Infants born at extremely low gestational age are at high risk for bronchopulmonary dysplasia and continuing lung disease. There are no early clinical biomarkers for pulmonary outcome and limited ...therapeutic interventions.
We performed global proteomics of premature infant tracheal aspirate (TA) and plasma to determine the composition and source of lung fluid proteins and to identify potential biomarkers of respiratory outcome.
TA samples were collected from intubated infants in the TOLSURF cohort before and after nitric oxide treatment, and plasma was collected from NO CLD infants. Protein abundance was assayed by HPLC/tandem mass spectrometry and Protein Prospector software. mRNA abundance in mid-gestation fetal lung was assessed by RNA sequencing. Pulmonary morbidity was defined as a need for ventilatory support at term and during the first year.
Abundant TA proteins included albumin, hemoglobin, and actin-related proteins. 96 of 137 detected plasma proteins were present in TA (r = 0.69, p<0.00001). Based on lung RNAseq data, ~88% of detected TA proteins in injured infant lung are derived at least in part from lung epithelium with overrepresentation in categories of cell membrane/secretion and stress/inflammation. Comparing 37 infants at study enrollment (7-14 days) who did or did not develop persistent pulmonary morbidity, candidate biomarkers of both lung (eg., annexin A5) and plasma (eg., vitamin D-binding protein) origin were identified. Notably, levels of free hemoglobin were 2.9-fold (p = 0.03) higher in infants with pulmonary morbidity. In time course studies, hemoglobin decreased markedly in most infants after enrollment coincident with initiation of inhaled nitric oxide treatment.
We conclude that both lung epithelium and plasma contribute to the lung fluid proteome in premature infants with lung injury. Early postnatal elevation of free hemoglobin and heme, which are both pro-oxidants, may contribute to persistent lung disease by depleting nitric oxide and increasing oxidative/nitrative stress.
Lung inflammation in premature infants contributes to the development of bronchopulmonary dysplasia (BPD), a chronic lung disease with long-term sequelae. Pilot studies administering budesonide ...suspended in surfactant have found reduced BPD without the apparent adverse effects that occur with systemic dexamethasone therapy. Our objective was to determine budesonide potency, stability, and antiinflammatory effects in human fetal lung. We cultured explants of second-trimester fetal lung with budesonide or dexamethasone and used microscopy, immunoassays, RNA sequencing, liquid chromatography/tandem mass spectrometry, and pulsating bubble surfactometry. Budesonide suppressed secreted chemokines IL-8 and CCL2 (MCP-1) within 4 hours, reaching a 90% decrease at 12 hours, which was fully reversed 72 hours after removal of the steroid. Half-maximal effects occurred at 0.04-0.05 nM, representing a fivefold greater potency than for dexamethasone. Budesonide significantly induced 3.6% and repressed 2.8% of 14,500 sequenced mRNAs by 1.6- to 95-fold, including 119 genes that contribute to the glucocorticoid inflammatory transcriptome; some are known targets of nuclear factor-κB. By global proteomics, 22 secreted inflammatory proteins were hormonally regulated. Two glucocorticoid-regulated genes of interest because of their association with lung disease are CHI3L1 and IL1RL1. Budesonide retained activity in the presence of surfactant and did not alter its surface properties. There was some formation of palmitate-budesonide in lung tissue but no detectable metabolism to inactive 16α-hydroxy prednisolone. We concluded that budesonide is a potent and stable antiinflammatory glucocorticoid in human fetal lung in vitro, supporting a beneficial antiinflammatory response to lung-targeted budesonide:surfactant treatment of infants for the prevention of BPD.
Total Parenteral Nutrition (TPN), which uses intravenous administration of nutrients, minerals and vitamins, is essential for sustaining premature infants until they transition to enteral feeds, but ...there is limited information on metabolomic differences between infants on TPN and enteral feeds. We performed untargeted global metabolomics on urine samples collected between 23-30 days of life from 314 infants born <29 weeks gestational age from the TOLSURF and PROP cohorts. Principal component analysis across all metabolites showed a separation of infants solely on TPN compared to infants who had transitioned to enteral feeds, indicating global metabolomic differences between infants based on feeding status. Among 913 metabolites that passed quality control filters, 609 varied in abundance between infants on TPN vs. enteral feeds at
< 0.05. Of these, 88% were in the direction of higher abundance in the urine of infants on enteral feeds. In a subset of infants in a longitudinal analysis, both concurrent and delayed changes in metabolite levels were observed with the initiation of enteral feeds. These infants had higher concentrations of essential amino acids, lipids, and vitamins, which are necessary for growth and development, suggesting the nutritional benefit of an enteral feeding regimen.
We have developed a transgenic mouse expressing enhanced green fluorescent protein (EGFP) in virtually all type II (TII) alveolar epithelial cells. The CBG mouse (SPC-BAC-EGFP) contains a bacterial ...artificial chromosome modified to express EGFP within the mouse surfactant protein (SP)-C gene 3' untranslated region. EGFP mRNA expression is limited to the lung. EGFP fluorescence is both limited to and exhibited by all cells expressing pro-SP-C; fluorescence is uniform throughout all lobes of the lung and does not change as mice age. EGFP(+) cells also express SP-B but do not express podoplanin, a type I (TI) cell marker. CBG mice show no evidence of lung disease with aging. In 3 hours, TII cells can be isolated in >99% purity from CBG mice by FACS; the yield of 3.7 ± 0.6 × 10(6) cells represents approximately 25 to 60% of the TII cells in the lung. By FACS analysis, approximately 0.9% of TII cells are in mitosis in uninjured lungs; after bleomycin injury, 4.1% are in mitosis. Because EGFP fluorescence can be detected for >14 days in culture, at a time that SP-C mRNA expression is essentially nil, this line may be useful for tracking TII cells in culture and in vivo. When CBG mice are crossed to transgenic mice expressing rat podoplanin, TI and TII cells can be easily simultaneously identified and isolated. When bred to other strains of mice, EGFP expression can be used to identify TII cells without the need for immunostaining for SP-C. These mice should be useful in models of mouse pulmonary disease and in studies of TII cell biology, biochemistry, and genetics.
Background Vascular endothelial growth factor (VEGF) is required for blood vessel formation during lung growth and repair. Alteration of VEGF isoform expression has been demonstrated in response to ...fetal tracheal occlusion and in models of lung injury. The purpose of this study was to investigate VEGF expression during compensatory lung growth in the mouse. Methods Under general anesthesia, adult mice underwent left thoracotomy with ( n = 5) or without (sham, n = 5) pneumonectomy. The right lungs were harvested at 1, 3, and 7 d after the operation. Lung-to-body weight ratio as well as total DNA and protein content were measured. VEGF protein expression was analyzed by Western blot and ELISA. VEGF isoform expression was evaluated using semi-quantitative PCR followed by Imagequant optical densitometry. Values were compared by Student's t -test and ANOVA using Fisher's protected least significant difference post-hoc test where appropriate. Results Compensatory lung growth was observed as measured by increases in right lung-to-body weight ratio and in DNA and protein content. Total VEGF RNA and protein expression did not change after pneumonectomy. However, on post-operative day 1, there was a decrease in the relative percentage of VEGF188 mRNA (P < 0.01), and an increase in the relative percentage of VEGF164 mRNA ( P = 0.05). At 3 d postpneumonectomy, low relative VEGF188 expression persisted ( P < 0.05), VEGF164 expression normalized, and relative VEGF120 expression increased ( P < 0.01). Isoform expression in the pneumonectomy animals was identical to sham animals by the seventh d. There were no differences observed in VEGF receptor expression. Conclusion During compensatory lung growth, we have observed an early postoperative reversion of VEGF isoform expression to the pattern seen during fetal lung development and in lung injury models.
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