The hydrocortisone-responsive urinary metabolome of premature infants.

BACKGROUND: Extremely premature infants are at risk for circulatory collapse or respiratory failure that are often treated with hydrocortisone (HC); however, there is no information on the metabolic consequences of this therapy. METHODS: Longitudinal urine samples from infants <28 weeks gestation in the Trial of Late Surfactant were analyzed by untargeted UHPLC:MS/MS. Fourteen infants who received a tapering course of HC beginning at 3 mg/kg/day for ≥9 days were compared to 14 matched control infants. A secondary cross-sectional analysis by logistic regression used urines from 314 infants. RESULTS: Of 1145 urinary metabolites detected, abundance of 219, representing all the major biochemical pathways, changed at p < 0.05 in the HC-treated group with 90% decreasing; 3 cortisol derivatives increased ~2-fold with HC therapy. Only 11% of regulated metabolites remained responsive at the lowest HC dose. Regulated metabolites included two steroids and thiamin that are associated with lung inflammation in infants. HC responsiveness was confirmed in 57% of metabolites by cross-sectional analysis. CONCLUSIONS: HC treatment of premature infants influenced in a dose-dependent manner abundance of 19% of identified urinary metabolites of diverse biochemical systems, primarily reducing concentrations. These findings indicate that exposure to HC reversibly impacts the nutritional status of premature infants. IMPACT: Hydrocortisone treatment of premature infants with respiratory failure or circulatory collapse alters levels of a subset of urinary metabolites representing all major biochemical pathways. This is the first description of the scope, magnitude, timing and reversibility of metabolomic changes in infants in response to hydrocortisone, and it confirms corticosteroid regulation of three biochemicals that are associated with lung inflammatory status. The findings indicate a dose-dependency of hydrocortisone for metabolomic and anti-inflammatory effects, that prolonged therapy may lower the supply of many nutrients, and that monitoring concentrations of cortisol and inflammation markers may be a useful clinical approach during corticosteroid therapy.

Blood metabolomics in infants enrolled in a dose escalation pilot trial of budesonide in surfactant.

BACKGROUND: The pathogenesis of BPD includes inflammation and oxidative stress in the immature lung. Corticosteroids improve respiratory status and outcome, but the optimal treatment regimen for benefit with low systemic effects is uncertain. METHODS: In a pilot dose escalation trial, we administered ≤5 daily doses of budesonide in surfactant to 24 intubated premature infants (Steroid And Surfactant in ELGANs (SASSIE)). Untargeted metabolomics was performed on dried blood spots using UPLC-MS/MS. Tracheal aspirate IL-8 concentration was determined as a measure of lung inflammation. RESULTS: Metabolomics data for 829 biochemicals were obtained on 121 blood samples over 96 h from 23 infants receiving 0.025, 0.05, or 0.1 mg budesonide/kg. Ninety metabolites were increased or decreased in a time- and dose-dependent manner at q ≤ 0.1 with overrepresentation in lipid and amino acid super pathways. Different dose response patterns occurred, with negative regulation associated with highest sensitivity to budesonide. Baseline levels of 22 regulated biochemicals correlated with lung inflammation (IL-8), with highest significance for sphingosine and thiamin. CONCLUSIONS: Numerous metabolic pathways are regulated in a dose-dependent manner by glucocorticoids, which apparently act via distinct mechanisms that impact dose sensitivity. The findings identify candidate blood biochemicals as biomarkers of lung inflammation and systemic responses to corticosteroids. IMPACT: Treatment of premature infants in respiratory failure with 0.1 mg/kg intra-tracheal budesonide in surfactant alters levels of ~11% of detected blood biochemicals in discrete time- and dose-dependent patterns. A subset of glucocorticoid-regulated biochemicals is associated with lung inflammatory status as assessed by lung fluid cytokine concentration. Lower doses of budesonide in surfactant than currently used may provide adequate anti-inflammatory responses in the lung with fewer systemic effects, improving the benefit:risk ratio.

Dose-escalation trial of budesonide in surfactant for prevention of bronchopulmonary dysplasia in extremely low gestational age high-risk newborns (SASSIE).

BACKGROUND: Initial trials of lung-targeted budesonide (0.25 mg/kg) in surfactant to prevent bronchopulmonary dysplasia (BPD) in premature infants have shown benefit; however, the optimal safe dose is unknown. METHODS: Dose-escalation study of budesonide (0.025, 0.05, 0.10 mg/kg) in calfactatant in extremely low gestational age neonates (ELGANs) requiring intubation at 3-14 days. Tracheal aspirate (TA) cytokines, blood budesonide concentrations, and untargeted blood metabolomics were measured. Outcomes were compared with matched infants receiving surfactant in the Trial Of Late SURFactant (TOLSURF). RESULTS: Twenty-four infants with mean gestational age 25.0 weeks and 743 g birth weight requiring mechanical ventilation were enrolled at mean age 6 days. Budesonide was detected in the blood of all infants with a half-life of 3.4 h. Of 11 infants with elevated TA cytokine levels at baseline, treatment was associated with sustained decrease (mean 65%) at all three dosing levels. There were time- and dose-dependent decreases in blood cortisol concentrations and changes in total blood metabolites. Respiratory outcomes did not differ from the historic controls. CONCLUSIONS: Budesonide/surfactant had no clinical respiratory benefit at any dosing levels for intubated ELGANs. One-tenth the dose used in previous trials had minimal systemic metabolic effects and appeared effective for lung-targeted anti-inflammatory action.

Cell fate analysis in fetal mouse lung reveals distinct pathways for TI and TII cell development.

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.

Surfactant status and respiratory outcome in premature infants receiving late surfactant treatment.

BACKGROUND: Many premature infants with respiratory failure are deficient in surfactant, but the relationship to occurrence of bronchopulmonary dysplasia (BPD) is uncertain. METHODS: Tracheal aspirates were collected from 209 treated and control infants enrolled at 7-14 days in the Trial of Late Surfactant. The content of phospholipid, surfactant protein B, and total protein were determined in large aggregate (active) surfactant. RESULTS: At 24 h, surfactant treatment transiently increased surfactant protein B content (70%, p < 0.01), but did not affect recovered airway surfactant or total protein/phospholipid. The level of recovered surfactant during dosing was directly associated with content of surfactant protein B (r = 0.50, p < 0.00001) and inversely related to total protein (r = 0.39, p < 0.0001). For all infants, occurrence of BPD was associated with lower levels of recovered large aggregate surfactant, higher protein content, and lower SP-B levels. Tracheal aspirates with lower amounts of recovered surfactant had an increased proportion of small vesicle (inactive) surfactant. CONCLUSIONS: We conclude that many intubated premature infants are deficient in active surfactant, in part due to increased intra-alveolar metabolism, low SP-B content, and protein inhibition, and that the severity of this deficit is predictive of BPD. Late surfactant treatment at the frequency used did not provide a sustained increase in airway surfactant.

Analysis of the microRNA signature driving adaptive right ventricular hypertrophy in an ovine model of congenital heart disease.

The right ventricular (RV) response to pulmonary arterial hypertension (PAH) is heterogeneous. Most patients have maladaptive changes with RV dilation and RV failure, whereas some, especially patients with PAH secondary to congenital heart disease, have an adaptive response with hypertrophy and preserved systolic function. Mechanisms for RV adaptation to PAH are unknown, despite RV function being a primary determinant of mortality. In our congenital heart disease ovine model with fetally implanted aortopulmonary shunt (shunt lambs), we previously demonstrated an adaptive physiological RV response to increased afterload with hypertrophy. In the present study, we examined small noncoding microRNA (miRNA) expression in shunt RV and characterized downstream effects of a key miRNA. RV tissue was harvested from 4-wk-old shunt and control lambs ( n = 5), and miRNA, mRNA, and protein were quantitated. We found differential expression of 40 cardiovascular-specific miRNAs in shunt RV. Interestingly, this miRNA signature is distinct from models of RV failure, suggesting that miRNAs might contribute to adaptive RV hypertrophy. Among RV miRNAs, miR-199b was decreased in the RV with eventual downregulation of nuclear factor of activated T cells/calcineurin signaling. Furthermore, antifibrotic miR-29a was increased in the shunt RV with a reduction of the miR-29 targets collagen type A1 and type 3A1 and decreased fibrosis. Thus, we conclude that the miRNA signature specific to shunt lambs is distinct from RV failure and drives gene expression required for adaptive RV hypertrophy. We propose that the adaptive RV miRNA signature may serve as a prognostic and therapeutic tool in patients with PAH to attenuate or prevent progression of RV failure and premature death. NEW & NOTEWORTHY This study describes a novel microRNA signature of adaptive right ventricular hypertrophy, with particular attention to miR-199b and miR-29a.

Antiinflammatory Effects of Budesonide in Human Fetal Lung.

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.

High-efficiency type II cell-enhanced green fluorescent protein expression facilitates cellular identification, tracking, and isolation.

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.

Surface film formation in vitro by infant and therapeutic surfactants: role of surfactant protein B.

BACKGROUND: Pulmonary surfactant provides an alveolar surface-active film that is critical for normal lung function. Our objective was to determine in vitro film formation properties of therapeutic and infant surfactants and the influence of surfactant protein (SP)-B content. METHODS: We used a multiwell fluorescent assay measuring maximum phospholipid surface accumulation (Max), phospholipid concentration required for half-maximal film formation (½Max), and time for maximal accumulation (tMax). RESULTS: Among five therapeutic surfactants, calfactant (highest SP-B content) had film formation values similar to natural surfactant, and addition of SP-B to beractant (lowest SP-B) normalized its Max value. Addition of budesonide to calfactant did not adversely affect film formation. In tracheal aspirates of preterm infants with evolving chronic lung disease, SP-B content correlated with ½Max and tMax values, and SP-B supplementation of SP-B-deficient infant surfactant restored normal film formation. Reconstitution of normal surfactant indicated a role for both SP-B and SP-C in film formation. CONCLUSION: Film formation in vitro differs among therapeutic surfactants and is highly dependent on SP-B content in infant surfactant. The results support a critical role of SP-B for promoting surface film formation.

Claudin-18 deficiency results in alveolar barrier dysfunction and impaired alveologenesis in mice.

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.

The Urinary Metabolomic Fingerprint in Extremely Preterm Infants on Total Parenteral Nutrition vs. Enteral Feeds.

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 p < 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.

Distribution and surfactant association of carcinoembryonic cell adhesion molecule 6 in human lung.

Carcinoembryonic cell adhesion molecule 6 (CEACAM6) is a glycosylated, glycophosphatidylinositol-anchored protein expressed in epithelial cells of various primate tissues. It binds gram-negative bacteria and is overexpressed in human cancers. CEACAM6 is associated with lamellar bodies of cultured type II cells of human fetal lung and protects surfactant function in vitro. In this study, we characterized CEACAM6 expression in vivo in human lung. CEACAM6 was present in lung lavage of premature infants at birth and increased progressively in intubated infants with lung disease. Of surfactant-associated CEACAM6, ∼80% was the fully glycosylated, 90-kDa form that contains the glycophosphatidylinositol anchor, and the concentration (3.9% of phospholipid for adult lung) was comparable to that for surfactant proteins (SP)-A/B/C. We examined the affinity of CEACAM6 by purification of surfactant on density gradient centrifugation; concentrations of CEACAM6 and SP-B per phospholipid were unchanged, whereas levels of total protein and SP-A decreased by 60%. CEACAM6 mRNA content decreased progressively from upper trachea to peripheral fetal lung, whereas protein levels were similar in all regions of adult lung, suggesting proximal-to-distal developmental expression in lung epithelium. In adult lung, most type I cells and ∼50% of type II cells were immunopositive. We conclude that CEACAM6 is expressed by alveolar and airway epithelial cells of human lung and is secreted into lung-lining fluid, where fully glycosylated protein binds to surfactant. Production appears to be upregulated during neonatal lung disease, perhaps related to roles of CEACAM6 in surfactant function, cell proliferation, and innate immune defense.

Regulated gene expression in cultured type II cells of adult human lung.

Alveolar type II cells have multiple functions, including surfactant production and fluid clearance, which are critical for lung function. Differentiation of type II cells occurs in cultured fetal lung epithelial cells treated with dexamethasone plus cAMP and isobutylmethylxanthine (DCI) and involves increased expression of 388 genes. In this study, type II cells of human adult lung were isolated at approximately 95% purity, and gene expression was determined (Affymetrix) before and after culturing 5 days on collagen-coated dishes with or without DCI for the final 3 days. In freshly isolated cells, highly expressed genes included SFTPA/B/C, SCGB1A, IL8, CXCL2, and SFN in addition to ubiquitously expressed genes. Transcript abundance was correlated between fetal and adult cells (r = 0.88), with a subset of 187 genes primarily related to inflammation and immunity that were expressed >10-fold higher in adult cells. During control culture, expression increased for 8.1% of expressed genes and decreased for approximately 4% including 118 immune response and 10 surfactant-related genes. DCI treatment promoted lamellar body production and increased expression of approximately 3% of probed genes by > or =1.5-fold; 40% of these were also induced in fetal cells. Highly induced genes (> or =10-fold) included PGC, ZBTB16, DUOX1, PLUNC, CIT, and CRTAC1. Twenty-five induced genes, including six genes related to surfactant (SFTPA/B/C, PGC, CEBPD, and ADFP), also had decreased expression during control culture and thus are candidates for hormonal regulation in vivo. Our results further define the adult human type II cell molecular phenotype and demonstrate that a subset of genes remains hormone responsive in cultured adult cells.

Vascular endothelial growth factor isoform and receptor expression during compensatory lung growth.

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.

Composition and origin of lung fluid proteome in premature infants and relationship to respiratory outcome.

BACKGROUND: 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. OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Expression of human carcinoembryonic antigen-related cell adhesion molecule 6 and alveolar progenitor cells in normal and injured lungs of transgenic mice.

Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is expressed in the epithelium of various primate tissues, including lung airway and alveoli. In human lung, CEACAM6 is developmentally and hormonally regulated, protects surfactant function, has anti-apoptotic activity and is dysregulated in cancers. We hypothesized that alveolar CEACAM6 expression increases in lung injury and promotes cell proliferation during repair. Studies were performed in CEABAC transgenic mice-containing human CEACAM genes. The level of CEACAM6 in adult CEABAC lung was comparable to that in human infants; expression occurred in epithelium of airways and of some alveoli but rarely co-localized with markers of type I or type II cells. Ten days after bleomycin instillation, both the number of CEACAM6(+) cells and immunostaining intensity were elevated in injured lung areas, and there was increased co-localization with type I and II cell markers. To specifically address type II cells, we crossed CEABAC mice with animals expressing EGFP driven by the SP-C promoter. After bleomycin injury, partially flattened, elongated epithelial cells were observed that expressed type I cell markers and were primarily either EGFP(+) or CEACAM6(+). In cell cycle studies, mitosis was greater in CEACAM6(+) non-type II cells versus CEACAM6(+)/EGFP(+) cells. CEACAM6 epithelial expression was also increased after hyperoxic exposure and LPS instillation, suggesting a generalized response to acute lung injuries. We conclude that CEACAM6 expression is comparable in human lung and the CEABAC mouse. CEACAM6 in this model appears to be a marker of a progenitor cell population that contributes to alveolar epithelial cell replenishment after lung injury.

A small-molecule smoothened agonist prevents glucocorticoid-induced neonatal cerebellar injury.

Glucocorticoids are used for treating preterm neonatal infants suffering from life-threatening lung, airway, and cardiovascular conditions. However, several studies have raised concerns about detrimental effects of postnatal glucocorticoid administration on the developing brain leading to cognitive impairment, cerebral palsy, and hypoplasia of the cerebellum, a brain region critical for coordination of movement and higher-order neurological functions. Previously, we showed that glucocorticoids inhibit Sonic hedgehog-Smoothened (Shh-Smo) signaling, the major mitogenic pathway for cerebellar granule neuron precursors. Conversely, activation of Shh-Smo in transgenic mice protects against glucocorticoid-induced neurotoxic effects through induction of the 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2) pathway. Here, we show that systemic administration of a small-molecule agonist of the Shh-Smo pathway (SAG) prevented the neurotoxic effects of glucocorticoids. SAG did not interfere with the beneficial effects of glucocorticoids on lung maturation, and despite the known associations of the Shh pathway with neoplasia, we found that transient (1-week-long) SAG treatment of neonatal animals was well tolerated and did not promote tumor formation. These findings suggest that a small-molecule agonist of Smo has potential as a neuroprotective agent in neonates at risk for glucocorticoid-induced neonatal cerebellar injury.

Dynamic tracheal occlusion improves lung morphometrics and function in the fetal lamb model of congenital diaphragmatic hernia.

BACKGROUND: Congenital diaphragmatic hernia (CDH) is associated with significant neonatal morbidity and mortality. Although prenatal complete tracheal occlusion (cTO) causes hypoplastic CDH lungs to enlarge, improved lung function has not been demonstrated. Furthermore, cTO interferes with the dynamic pressure change and fluid flow associated with fetal breathing. PURPOSE: The purpose of the study was to assess a novel dynamic tracheal occlusion (dTO) device that preserves pressure changes and fluid flow. METHODS: In this pilot study, CDH was created in fetal lambs at 65 days of gestational age (GA). At 110 days GA, a cTO device (n = 3) or a dTO device (n = 4) was placed in the fetal trachea. At 135 days GA, lambs were delivered and resuscitated. Unoperated lamb co-twins (n = 5), sham thoracotomy lambs (n = 2), and untreated CDH lambs (n = 3) served as controls. RESULTS: Tracheal opening pressure, lung volume, lung fluid total protein, and phospholipid were significantly higher in the cTO group than in the dTO and unoperated control groups. Maximal oxygenation and lung compliance were significantly lower in the cTO group when compared with the unoperated control and dTO groups. CONCLUSION: Preliminary results suggest that in the fetal lamb CDH model, dTO restores normal lung morphometrics and function, whereas cTO leads to enlarged but less functional lungs.

Congenital diaphragmatic hernia prevents absorption of distal air space fluid in late-gestation rat fetuses.

We hypothesized that congenital diaphragmatic hernia (CDH) may decrease distal air space fluid absorption due to immaturity of alveolar epithelial cells from a loss of the normal epithelial Na+ transport, as assessed by amiloride and epithelial Na+ channel (ENaC) and Na-K-ATPase expression, as well as failure to respond to endogenous epinephrine as assessed by propranolol. Timed-pregnant dams were gavage fed 100 mg of nitrofen at 9.5-day gestation to induce CDH in the fetuses, and distal air space fluid absorption experiments were carried out on 22-day gestation (term) fetuses. Controls were nitrofen-exposed fetuses without CDH. Absorption of distal air space fluid was measured from the increase in 131I-albumin concentration in an isosmolar, physiological solution instilled into the developing lungs. In controls, distal air space fluid absorption was rapid and mediated by beta-adrenoceptors as demonstrated by reversal to fluid secretion after propranolol. Normal lung fluid absorption was also partially inhibited by amiloride. In contrast, CDH fetuses continued to show lung fluid secretion, and this secretion was not affected by either propranolol or amiloride. CDH lungs showed a 67% reduction in alpha-ENaC and beta-ENaC expression, but no change in alpha1-Na-K-ATPase expression. These studies demonstrate: 1) CDH delays lung maturation with impaired distal air space fluid absorption secondary to inadequate Na+ uptake by the distal lung epithelium that results in fluid-filled lungs at birth with reduced capacity to establish postnatal breathing, and 2) the main stimulus to lung fluid absorption in near-term control fetuses, elevated endogenous epinephrine levels, is not functional in CDH fetuses.

Changes in fetal lung distension alter expression of vascular endothelial growth factor and its isoforms in developing rat lung.

Vascular endothelial growth factor A (VEGF-A) is essential for normal pulmonary vascular and parenchymal development. Changes in fetal lung distension profoundly affect lung growth and maturation, including vascular development. To define developmental lung expression of VEGF-A and its receptors and investigate effects of changes in fetal lung distension, we studied fetal rats at embryonic day (ED) 16, 19, and 22, postnatal rats at postnatal day (PD) 5, 10, and 21, and adult rats. We used reverse transcriptase PCR to measure mRNA expression for VEGF-A isoforms (VEGF-A(120), (-144), (-164), and (-188)) and VEGF-A receptors, Flt-1 and Flk-1. With advancing development, mRNA content increased only for VEGF-A(188) (p < 0.05) and for Flt-1 (p < 0.02) and Flk-1 (p < 0.005). As a percentage of total VEGF-A mRNA, VEGF-A(188) (15% at ED 16) increased to become the dominant isoform at PD 21 (40%, p < 0.005) and adulthood; in contrast, there were decreases in both VEGF-A(144) (p < 0.05) and (-120) (p < 0.005). VEGF-A protein was expressed in alveolar epithelium (type I and II cells) and interstitium. Increasing fetal lung distension by tracheal occlusion (TO) accelerated the normal maturational pattern of VEGF-A isoforms and increased VEGF-A protein; decreasing fetal lung distension by congenital diaphragmatic hernia (CDH) retarded the normal developmental pattern and decreased VEGF-A protein. Neither TO nor CDH consistently affected Flt-1 or Flk-1 mRNA content. These results show that mechanical factors significantly affect lung VEGF-A expression and suggest that VEGF-A mediates previously described changes in lung vascular and parenchymal development caused by CDH and by TO.