Tracheal Aspirates Of Preterm Infants Research Articles

Hypoxia Inducible Factor-1α in SM22α Expressing Cells Modulates Alveolarization.

Worldwide, the incidence of both preterm births and chronic lung disease of infancy, or bronchopulmonary dysplasia (BPD), remains high. Infants with BPD have larger and fewer alveoli, pathology that can persist into adulthood. Though hypoxia inducible factor-1α (HIF-1α) plays a significant role in mediating pulmonary angiogenesis and alveolarization, the cell specific role of HIF-1α remains incompletely understood. To determine whether HIF-1α, in a mesenchymal cells (MC) subset, mediates postnatal alveolarization. We generated mice with cell-specific deletion of HIF-1α by crossing SM22α-promoter-driven Cre mice with HIF-1αflox/flox mice (SM22α- HIF-1α-/-), determined SM22α-expressing cell identity using single cell RNA-seq, and interrogated clinical samples from preterm infants. Measurement and Main Outcomes: Deletion of HIF-1α in SM22α expressing cells had no effect on lung structure at day (d) 3 of life. However, at 8d there were fewer and larger alveoli, a difference that persisted into adulthood. Microvascular density, elastin organization, and peripheral branching of the lung vasculature were decreased in SM22α-HIF-1α-/-, compared to control, mice. Single cell RNA-seq demonstrated that 3 MC subtypes (myofibroblasts, airway and vascular smooth muscle cells (VSMC)) express SM22α. Pulmonary VSMC from SM22α- HIF-1α-/- had decreased angiopoetin 2 expression and, in co-culture experiments, a diminished capacity to promote angiogenesis that was rescued by angiopoetin-2. Angiopoetin-2 expression in the tracheal aspirates of preterm infants was inversely correlated with overall time on mechanical ventilation, a marker of disease severity. SM22α-specific HIF-1α expression drives peripheral angiogenesis and alveolarization in the lung, perhaps by promoting angiopoetin-2 expression.

TRAIL protects the immature lung from hyperoxic injury

The hyperoxia-induced pro-inflammatory response and tissue damage constitute pivotal steps leading to bronchopulmonary dysplasia (BPD) in the immature lung. The pro-inflammatory cytokines are considered attractive candidates for a directed intervention but the complex interplay between inflammatory and developmental signaling pathways requires a comprehensive evaluation before introduction into clinical trials as studied here for the death inducing ligand TRAIL. At birth and during prolonged exposure to oxygen and mechanical ventilation, levels of TRAIL were lower in tracheal aspirates of preterm infants <29 weeks of gestation which developed moderate/severe BPD. These findings were reproduced in the newborn mouse model of hyperoxic injury. The loss of TRAIL was associated with increased inflammation, apoptosis induction and more pronounced lung structural simplification after hyperoxia exposure for 7 days while activation of NFκB signaling during exposure to hyperoxia was abrogated. Pretreatment with recombinant TRAIL rescued the developmental distortions in precision cut lung slices of both wildtype and TRAIL−/− mice exposed to hyperoxia. Of importance, TRAIL preserved alveolar type II cells, mesenchymal progenitor cells and vascular endothelial cells. In the situation of TRAIL depletion, our data ascribe oxygen toxicity a more injurious impact on structural lung development. These data are not surprising taking into account the diverse functions of TRAIL and its stimulatory effects on NFκB signaling as central driver of survival and development. TRAIL exerts a protective role in the immature lung as observed for the death inducing ligand TNF-α before.

The Role of Sphingolipid Signaling in Oxidative Lung Injury and Pathogenesis of Bronchopulmonary Dysplasia.

Premature infants are born with developing lungs burdened by surfactant deficiency and a dearth of antioxidant defense systems. Survival rate of such infants has significantly improved due to advances in care involving mechanical ventilation and oxygen supplementation. However, a significant subset of such survivors develops the chronic lung disease, Bronchopulmonary dysplasia (BPD), characterized by enlarged, simplified alveoli and deformed airways. Among a host of factors contributing to the pathogenesis is oxidative damage induced by exposure of the developing lungs to hyperoxia. Recent data indicate that hyperoxia induces aberrant sphingolipid signaling, leading to mitochondrial dysfunction and abnormal reactive oxygen species (ROS) formation (ROS). The role of sphingolipids such as ceramides and sphingosine 1-phosphate (S1P), in the development of BPD emerged in the last decade. Both ceramide and S1P are elevated in tracheal aspirates of premature infants of <32 weeks gestational age developing BPD. This was faithfully reflected in the murine models of hyperoxia and BPD, where there is an increased expression of sphingolipid metabolites both in lung tissue and bronchoalveolar lavage. Treatment of neonatal pups with a sphingosine kinase1 specific inhibitor, PF543, resulted in protection against BPD as neonates, accompanied by improved lung function and reduced airway remodeling as adults. This was accompanied by reduced mitochondrial ROS formation. S1P receptor1 induced by hyperoxia also aggravates BPD, revealing another potential druggable target in this pathway for BPD. In this review we aim to provide a detailed description on the role played by sphingolipid signaling in hyperoxia induced lung injury and BPD.

MicroRNA Signatures Associated with Bronchopulmonary Dysplasia Severity in Tracheal Aspirates of Preterm Infants.

Bronchopulmonary dysplasia (BPD) is a form of chronic lung disease that develops in neonates as a consequence of preterm birth, arrested fetal lung development, and inflammation. The incidence of BPD remains on the rise as a result of increasing survival of extremely preterm infants. Severe BPD contributes to significant health care costs and is associated with prolonged hospitalizations, respiratory infections, and neurodevelopmental deficits. In this study, we aimed to detect novel biomarkers of BPD severity. We collected tracheal aspirates (TAs) from preterm babies with mild/moderate (n = 8) and severe (n = 17) BPD, and we profiled the expression of 1048 miRNAs using a PCR array. Associations with biological pathways were determined with the Ingenuity Pathway Analysis (IPA) software. We found 31 miRNAs differentially expressed between the two disease groups (2-fold change, false discovery rate (FDR) < 0.05). Of these, 4 miRNAs displayed significantly higher expression levels, and 27 miRNAs had significantly lower expression levels in the severe BPD group when compared to the mild/moderate BPD group. IPA identified cell signaling and inflammation pathways associated with miRNA signatures. We conclude that TAs of extremely premature infants contain miRNA signatures associated with severe BPD. These may serve as potential biomarkers of disease severity in infants with BPD.

Neutrophilic inflammation during lung development disrupts elastin assembly and predisposes adult mice to COPD.

Emerging evidence indicates that early life events can increase the risk for developing chronic obstructive pulmonary disease (COPD). Using an inducible transgenic mouse model for NF-κB activation in the airway epithelium, we found that a brief period of inflammation during the saccular stage (P3-P5) but not alveolar stage (P10-P12) of lung development disrupted elastic fiber assembly, resulting in permanent reduction in lung function and development of a COPD-like lung phenotype that progressed through 24 months of age. Neutrophil depletion prevented disruption of elastic fiber assembly and restored normal lung development. Mechanistic studies uncovered a role for neutrophil elastase (NE) in downregulating expression of critical elastic fiber assembly components, particularly fibulin-5 and elastin. Further, purified human NE and NE-containing exosomes from tracheal aspirates of premature infants with lung inflammation downregulated elastin and fibulin-5 expression by saccular-stage mouse lung fibroblasts. Together, our studies define a critical developmental window for assembling the elastin scaffold in the distal lung, which is required to support lung structure and function throughout the lifespan. Although neutrophils play a well-recognized role in COPD development in adults, neutrophilic inflammation may also contribute to early-life predisposition to COPD.

The protective effect of CXC chemokine receptor 2 antagonist on experimental bronchopulmonary dysplasia induced by postnatal systemic inflammation.

BackgroundAnimal studies have shown that a leukocyte influx precedes the development of bronchopulmonary dysplasia (BPD) in premature sheep. The CXC chemokine receptor 2 (CXCR2) pathway has been implicated in the pathogenesis of BPD because of the predominance of CXCR2 ligands in tracheal aspirates of preterm infants who later developed BPD.PurposeTo test the effect of CXCR2 antagonist on postnatal systemic and pulmonary inflammation and alveolarization in a newborn Sprague-Dawley rat model of BPD.MethodsLipopolysaccharide (LPS) was injected intraperitoneally (i.p.) into the newborn rats on postnatal day 1 (P1), P3, and P5 to induce systemic inflammation and inhibit alveolarization. In the same time with LPS administration, CXCR2 antagonist (SB-265610) or vehicle was injected i.p. to investigate whether CXCR2 antagonist can alleviate the detrimental effect of LPS on alveolarization by attenuating inflammation. On P7 and P14, bronchoalveolar lavage fluid (BALF) and peripheral blood (PB) were collected from the pups. To assess alveolarization, mean cord length and alveolar surface area were measured on 4 random nonoverlapping fields per animal in 2 distal lung sections at ×100 magnification.ResultsEarly postnatal LPS administration significantly increased neutrophil counts in BALF and PB and inhibited alveolarization, which was indicated by a greater mean cord length and lesser alveolar surface area. CXCR2 antagonist significantly attenuated the increase of neutrophil counts in BALF and PB and restored alveolarization as indicated by a decreased mean cord length and increased alveolar surface area in rat pups exposed to early postnatal systemic LPS.ConclusionCXCR2 antagonist preserved alveolarization by alleviating pulmonary and systemic inflammation induced by early postnatal systemic LPS administration. These results suggest that CXCR2 antagonist can be considered a potential therapeutic agent for BPD that results from disrupted alveolarization induced by inflammation.

Activation of the NF-κB pathway alters the phenotype of MSCs in the tracheal aspirates of preterm infants with severe BPD.

Mesenchymal stromal cells (MSCs) are released into the airways of preterm infants following lung injury. These cells display a proinflammatory phenotype and are associated with development of severe bronchopulmonary dysplasia (BPD). We aimed to characterize the functional properties of MSCs obtained from tracheal aspirates of 50 preterm infants who required invasive ventilation. Samples were separated by disease severity. The increased proliferative capacity of MSCs was associated with longer duration of mechanical ventilation and higher severity of BPD. Augmented growth depended on nuclear accumulation of NFκBp65 and was accompanied by reduced expression of cytosolic α-smooth muscle actin (α-SMA). The central role of NF-κB signaling was confirmed by inhibition of IκBα phosphorylation. The combined score of proliferative capacity, accumulation of NFκBp65, and expression of α-SMA was used to predict the development of severe BPD with an area under the curve (AUC) of 0.847. We mimicked the clinical situation in vitro, and stimulated MSCs with IL-1β and TNF-α. Both cytokines induced similar and persistent changes as was observed in MSCs obtained from preterm infants with severe BPD. RNA interference was employed to investigate the mechanistic link between NFκBp65 accumulation and alterations in phenotype. Our data indicate that determining the phenotype of resident pulmonary MSCs represents a promising biomarker-based approach. The persistent alterations in phenotype, observed in MSCs from preterm infants with severe BPD, were induced by the pulmonary inflammatory response. NFκBp65 accumulation was identified as a central regulatory mechanism. Future preclinical and clinical studies, aimed to prevent BPD, should focus on phenotype changes in pulmonary MSCs.

Ceramides in tracheal aspirates of preterm infants: Marker for bronchopulmonary dysplasia.

BackgroundIn an experimental mouse model we showed that ceramides play a role in the pathogenesis of bronchopulmonary dysplasia (BPD) and are a potential target for therapeutic intervention. We investigated whether ceramides are detectable in tracheal aspirates (TAs) of preterm infants and differ between infants with or without BPD.MethodsInfants born ≤ 32 weeks of gestational age in need of mechanical ventilation in the first week of life were included. TAs were obtained directly after intubation and at day 1, 3, 5, 7, and 14. Ceramide concentrations were measured by tandem mass spectrometry. At 36 weeks postmenstrual age BPD was defined as having had ≥ 28 days supplemental oxygen.Results122 infants were included, of which 14 died and 41 developed BPD. All infants showed an increase in ceramides after the first day of intubation. The ceramide profile differed significantly between preterm infants who did and did not develop BPD. However, the ceramide profile had no additional predictive value for BPD development over GA at birth, birth weight and total days of mechanical ventilation.ConclusionsCeramides are measurable in TAs of preterm born infants and may be an early marker for BPD development.

Inflammatory Mediators in Tracheal Aspirates of Preterm Infants Participating in a Randomized Trial of Permissive Hypercapnia.

Ventilator-induced lung injury is considered to be a main factor in the pathogenesis of bronchopulmonary dysplasia (BPD). Optimizing ventilator strategies may reduce respiratory morbidities in preterm infants. Permissive hypercapnia has been suggested to attenuate lung injury. We aimed to determine if a higher PCO2 target range results in less lung injury compared to the control target range and possibly reduces pro-inflammatory cytokines and acid sphingomyelinase (ASM) in tracheal aspirates (TA), which has not been addressed before. During a multicenter trial of permissive hypercapnia in extremely low birthweight infants (PHELBI), preterm infants (birthweight 400-1,000 g, gestational age 23 0/7-28 6/7 weeks) requiring mechanical ventilation within 24 h of birth were randomly assigned to a high PCO2 target or a control group. The high target group aimed at PCO2 values of 55-65, 60-70, and 65-75 mmHg and the control group at PCO2 values of 40-50, 45-55 and 50-60 mmHg on postnatal days 1-3, 4-6, and 7-14, respectively. TA was analyzed for pro-inflammatory cytokines from postnatal day 2-21. BPD was determined at a postmenstrual age of 36 weeks ± 2 days. Levels of inflammatory cytokines and ASM were similar in both groups: interleukin (IL)-6 (p = 0.14), IL-8 (p = 0.43), IL-10 (p = 0.24), IL-1β (p = 0.11), macrophage inflammatory protein 1α (p = 0.44), albumin (p = 0.41), neuropeptide Y (p = 0.52), leukotriene B4 (p = 0.11), transforming growth factor-β1 (p = 0.68), nitrite (p = 0.15), and ASM (p = 0.94). Furthermore, most inflammatory mediators were strongly affected by the age of the infants and increased from postnatal day 2 to 21. BPD or death was observed in 14 out of 62 infants, who were distributed evenly between both groups. The results suggest that high PCO2 target levels did not result in lower pulmonary inflammatory activity and thus reflect clinical results. This indicates that high PCO2 target ranges are not effective in reducing ventilator-induced lung injury in preterm infants, as compared to control targets. ISRCTN56143743.

Inflammatory Mediators in Tracheal Aspirates of Preterm Infants Participating in a Randomized Trial of Inhaled Nitric Oxide.

BackgroundVentilated preterm infants frequently develop bronchopulmonary dysplasia (BPD) which is associated with elevated inflammatory mediators in their tracheal aspirates (TA). In animal models of BPD, inhaled nitric oxide (iNO) has been shown to reduce lung inflammation, but data for human preterm infants is missing.MethodsWithin a European multicenter trial of NO inhalation for preterm infants to prevent BPD (EUNO), TA was collected to determine the effects of iNO on pulmonary inflammation. TA was collected from 43 premature infants randomly assigned to receive either iNO or placebo gas (birth weight 530–1230 g, median 800 g, gestational age 24 to 28 2/7 weeks, median 26 weeks). Interleukin (IL)-1β, IL-6, IL-8, transforming growth factor (TGF)-β1, interferon γ-induced protein 10 (IP-10), macrophage inflammatory protein (MIP)-1α, acid sphingomyelinase (ASM), neuropeptide Y and leukotriene B4 were measured in serial TA samples from postnatal day 2 to 14. Furthermore, TA levels of nitrotyrosine and nitrite were determined under iNO therapy.ResultsThe TA levels of IP-10, IL-6, IL-8, MIP-1α, IL-1β, ASM and albumin increased with advancing postnatal age in critically ill preterm infants, whereas nitrotyrosine TA levels declined in both, iNO-treated and placebo-treated infants. The iNO treatment generally increased nitrite TA levels, whereas nitrotyrosine TA levels were not affected by iNO treatment. Furthermore, iNO treatment transiently reduced early inflammatory and fibrotic markers associated with BPD development including TGF-β1, IP-10 and IL-8, but induced a delayed increase of ASM TA levels.ConclusionTreatment with iNO may have played a role in reducing several inflammatory and fibrotic mediators in TA of preterm infants compared to placebo-treated infants. However, survival without BPD was not affected in the main EUNO trial.Trial registrationNCT00551642

Molecular Detection of Ureaplasma Urealyticum and Ureaplasma Parvum Colonization in Preterm Infants with Respiratory Distress Syndrome

Background: Early neonatal acquisition of Ureaplasma urealyticum (U. urealyticum) and Ureaplasma parvum (U. parvum), in the 1 st 24 hours after birth and during passage of an infected maternal birth canal, have been linked to the development of respiratory diseases in premature newborns as a result of respiratory tract colonization. However, the role of these pathogens in the development of respiratory distress syndrome (RDS) still needs more clarification. Objectives: In this study we aimed to search for U. urealyticum and U. parvum in tracheal aspirates of premature infants with RDS by species-specific PCR assays and to evaluate the short term outcome of preterm infants with positive results. Methodology: Tracheal aspirate fluid samples from 35 preterm neonates with RDS with a mean gestational age of (32.76±2.48) weeks were collected within the first 24 hours after birth and analyzed for detection of U. urealyticum and U. parvum by species-specific PCR assays targeting urease gene subunits and adjoining spacer regions. Results: Ureaplasma spp. was detected in the tracheal aspirate of 6 (17%) preterm infants with RDS. Among these 6 positive cases; 5 (83%) were positive for U. parvum while only 1 case (17%) was positive for U. urealyticum. The preterm infants with positive results for Ureaplasma spp. had statistically significant higher degree of RD depending on the chest X ray grading results and longer durations of ventilation and hospitalization. Conclusions: Limited by the relatively small sample size, this work can be considered as a pilot study indicating a possible association between the severity of RDS in preterm infants and respiratory tract (RT) colonization with Ureaplasma spp. particularly U.parvum.

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

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. 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). 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. 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.

Reduced platelet-derived growth factor receptor expression is a primary feature of human bronchopulmonary dysplasia.

Animal studies have shown that platelet-derived growth factor (PDGF) signaling is required for normal alveolarization. Changes in PDGF receptor (PDGFR) expression in infants with bronchopulmonary dysplasia (BPD), a disease of hypoalveolarization, have not been examined. We hypothesized that PDGFR expression is reduced in neonatal lung mesenchymal stromal cells (MSCs) from infants who develop BPD. MSCs from tracheal aspirates of premature infants requiring mechanical ventilation in the first week of life were studied. MSC migration was assessed in a Boyden chamber. Human lung tissue was obtained from the University of Rochester Neonatal Lung Biorepository. Neonatal mice were exposed to air or 75% oxygen for 14 days. PDGFR expression was quantified by qPCR, immunoblotting, and stereology. MSCs were isolated from 25 neonates (mean gestational age 27.7 wk); 13 developed BPD and 12 did not. MSCs from infants who develop BPD showed lower PDGFR-α and PDGFR-β mRNA and protein expression and decreased migration to PDGF isoforms. Lungs from infants dying with BPD show thickened alveolar walls and paucity of PDGFR-α-positive cells in the dysmorphic alveolar septa. Similarly, lungs from hyperoxia-exposed neonatal mice showed lower expression of PDGFR-α and PDGFR-β, with significant reductions in the volume of PDGFR-α-positive alveolar tips. In conclusion, MSCs from infants who develop BPD hold stable alterations in PDGFR gene expression that favor hypoalveolarization. These data demonstrate that defective PDGFR signaling is a primary feature of human BPD.

Glycogen synthase kinase-3β/β-catenin signaling regulates neonatal lung mesenchymal stromal cell myofibroblastic differentiation

In bronchopulmonary dysplasia (BPD), alveolar septa are thickened with collagen and α-smooth muscle actin-, transforming growth factor (TGF)-β-positive myofibroblasts. We examined the biochemical mechanisms underlying myofibroblastic differentiation, focusing on the role of glycogen synthase kinase-3β (GSK-3β)/β-catenin signaling pathway. In the cytoplasm, β-catenin is phosphorylated on the NH(2) terminus by constitutively active GSK-3β, favoring its degradation. Upon TGF-β stimulation, GSK-3β is phosphorylated and inactivated, allowing β-catenin to translocate to the nucleus, where it activates transcription of genes involved in myofibroblastic differentiation. We examined the role of β-catenin in TGF-β1-induced myofibroblastic differentiation of neonatal lung mesenchymal stromal cells (MSCs) isolated from tracheal aspirates of premature infants with respiratory distress. TGF-β1 increased β-catenin expression and nuclear translocation. Transduction of cells with GSK-3β S9A, a nonphosphorylatable, constitutively active mutant that favors β-catenin degradation, blocked TGF-β1-induced myofibroblastic differentiation. Furthermore, transduction of MSCs with ΔN-catenin, a truncation mutant that cannot be phosphorylated on the NH(2) terminus by GSK-3β and is not degraded, was sufficient for myofibroblastic differentiation. In vivo, hyperoxic exposure of neonatal mice increases expression of β-catenin in α-smooth muscle actin-positive myofibroblasts. Similar changes were found in lungs of infants with BPD. Finally, low-passage unstimulated MSCs from infants developing BPD showed higher phospho-GSK-3β, β-catenin, and α-actin content compared with MSCs from infants not developing this disease, and phospho-GSK-3β and β-catenin each correlated with α-actin content. We conclude that phospho-GSK-3β/β-catenin signaling regulates α-smooth muscle actin expression, a marker of myofibroblast differentiation, in vitro and in vivo. This pathway appears to be activated in lung mesenchymal cells from patients with BPD.

Neonatal periostin knockout mice are protected from hyperoxia-induced alveolar simplication.

In bronchopulmonary dysplasia (BPD), alveolar septae are thickened with collagen and α-smooth muscle actin, transforming growth factor (TGF)-β-positive myofibroblasts. Periostin, a secreted extracellular matrix protein, is involved in TGF-β-mediated fibrosis and myofibroblast differentiation. We hypothesized that periostin expression is required for hypoalveolarization and interstitial fibrosis in hyperoxia-exposed neonatal mice, an animal model for this disease. We also examined periostin expression in neonatal lung mesenchymal stromal cells and lung tissue of hyperoxia-exposed neonatal mice and human infants with BPD. Two-to-three day-old wild-type and periostin null mice were exposed to air or 75% oxygen for 14 days. Mesenchymal stromal cells were isolated from tracheal aspirates of premature infants. Hyperoxic exposure of neonatal mice increased alveolar wall periostin expression, particularly in areas of interstitial thickening. Periostin co-localized with α-smooth muscle actin, suggesting synthesis by myofibroblasts. A similar pattern was found in lung sections of infants dying of BPD. Unlike wild-type mice, hyperoxia-exposed periostin null mice did not show larger air spaces or α-smooth muscle-positive myofibroblasts. Compared to hyperoxia-exposed wild-type mice, hyperoxia-exposed periostin null mice also showed reduced lung mRNA expression of α-smooth muscle actin, elastin, CXCL1, CXCL2 and CCL4. TGF-β treatment increased mesenchymal stromal cell periostin expression, and periostin treatment increased TGF-β-mediated DNA synthesis and myofibroblast differentiation. We conclude that periostin expression is increased in the lungs of hyperoxia-exposed neonatal mice and infants with BPD, and is required for hyperoxia-induced hypoalveolarization and interstitial fibrosis.

Isolation of Tracheal Aspirate Mesenchymal Stromal Cells Predicts Bronchopulmonary Dysplasia

We have isolated mesenchymal stem cells (MSCs) from tracheal aspirates of premature infants with respiratory distress. Under the influence of transforming growth factor β, MSCs differentiate into α-smooth-muscle actin-expressing myofibroblasts. Myofibroblasts are increased in the lungs of patients with bronchopulmonary dysplasia (BPD), a chronic lung disease of prematurely born infants. We tested whether isolation of MSCs from tracheal aspirates of premature infants with respiratory distress during the first week of life correlates with BPD. Eighty-four infants born at a gestational age of <33 weeks and requiring mechanical ventilation were studied. Aspirates were collected during suctioning and centrifuged. Cell pellets were resuspended in culture medium and plated. Adherent cells were grown to confluence. MSCs were isolated from the tracheal aspirates of 56 infants; 28 aspirate samples showed no MSCs. There was no statistical difference in gestational age or birth weight between the MSC and no-MSC groups. In the MSC group, 12 infants died and 25 developed BPD, as defined by a requirement for supplemental oxygen at 36 weeks' postmenstrual age. In the no-MSC group, 6 infants died and 1 developed BPD. Accounting for potential influences of gender, birth weight, gestational age, number of tracheal aspirate samples taken, and the duration of endotracheal intubation (up to 7 days), isolation of MSCs increased the adjusted odds ratio of BPD more than 21-fold (95% confidence interval: 1.82-265.85). Isolation of tracheal aspirate MSCs predicts the development of BPD, which suggests that MSCs play an important role in the pathogenesis of this disease.

Autocrine production of TGF-beta1 promotes myofibroblastic differentiation of neonatal lung mesenchymal stem cells.

We have isolated mesenchymal stem cells (MSCs) from tracheal aspirates of premature infants with respiratory distress. We examined the capacity of MSCs to differentiate into myofibroblasts, cells that participate in lung development, injury, and repair. Gene expression was measured by array, qPCR, immunoblot, and immunocytochemistry. Unstimulated MSCs expressed mRNAs encoding contractile (e.g., ACTA2, TAGLN), extracellular matrix (COL1A1 and ELN), and actin-binding (DBN1, PXN) proteins, consistent with a myofibroblast phenotype, although there was little translation into immunoreactive protein. Incubation in serum-free medium increased contractile protein (ACTA2, MYH11) gene expression. MSC-conditioned medium showed substantial levels of TGF-beta1, and treatment of serum-deprived cells with a type I activin receptor-like kinase inhibitor, SB-431542, attenuated the expression of genes encoding contractile and extracellular matrix proteins. Treatment of MSCs with TGF-beta1 further induced the expression of mRNAs encoding contractile (ACTA2, MYH11, TAGLN, DES) and extracellular matrix proteins (FN1, ELN, COL1A1, COL1A2), and increased the protein expression of alpha-smooth muscle actin, myosin heavy chain, and SM22. In contrast, human bone marrow-derived MSCs failed to undergo TGF-beta1-induced myofibroblastic differentiation. Finally, primary cells from tracheal aspirates behaved in an identical manner as later passage cells. We conclude that human neonatal lung MSCs demonstrate an mRNA expression pattern characteristic of myofibroblast progenitor cells. Autocrine production of TGF-beta1 further drives myofibroblastic differentiation, suggesting that, in the absence of other signals, fibrosis represents the "default program" for neonatal lung MSC gene expression. These data are consistent with the notion that MSCs play a key role in neonatal lung injury and repair.

Pulmonary Secretory Phospholipase A2 in Infants with Respiratory Distress Syndrome Stimulates in vitro Neutrophil Migration

Background: The massive pulmonary neutrophil influx in respiratory distress syndrome (RDS) in preterm infants has been ascribed to the effect of leukotriene B₄ (LTB₄). Objectives: To investigate whether secretory phospholipase A₂ (sPLA₂), the rate-limiting enzyme in LTB₄ production, is present in lungs of RDS infants and stimulates neutrophil migration. Methods: sPLA₂ was measured in tracheal aspirates from 15 preterm infants with RDS. The effect of aspirates on cord blood neutrophil migration was first measured, and the contribution of sPLA₂ was assessed by addition of its endogenous inhibitor Clara cell protein (CC16) or absorption of sPLA₂ from the aspirates. The role of intracellular signal transduction activation and LTB₄ formation in sPLA₂-induced neutrophil migration was determined using purified sPLA₂, several inhibitors of signal transduction, a LTB₄ synthesis inhibitor and a LTB₄ receptor antagonist. Results: All aspirates contained sPLA₂, which significantly stimulated neutrophil migration. Addition of CC16 or absorption of sPLA₂ abolished the stimulatory effect. All inhibitors significantly reduced sPLA₂-induced neutrophil migration. Conclusions: sPLA₂ is present in tracheal aspirates of preterm infants with RDS. Human recombinant sPLA₂ and pancreatic type sPLA₂ stimulate in vitro cord blood neutrophil migration via activation of intracellular signal transduction pathways, LTB₄ production and receptor binding. We speculate that sPLA₂ contributes to pulmonary neutrophil influx in RDS. Further studies are needed to determine the potential of sPLA₂ inhibition as a treatment for RDS.

Low levels of tissue inhibitors of metalloproteinases with a high matrix metalloproteinase-9/tissue inhibitor of metalloproteinase-1 ratio are present in tracheal aspirate fluids of infants who develop chronic lung disease.

The pathogenesis of chronic lung disease (CLD) involves inflammation with proteolytic damage to lung extracellular matrix. Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that, acting in concert with their tissue inhibitors, tightly orchestrate extracellular matrix morphogenesis and repair after injury. Imbalances in their levels relative to that of their inhibitors have been implicated in diseases characterized by matrix disruption and remodeling. We investigated the possibility that imbalances in MMP-9 and MMP-2 relative to their tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2, respectively, in tracheal aspirates of preterm infants may be involved in the development of CLD. Serial tracheal aspirates collected from birth until extubation in 49 ventilated preterm infants (24-32 weeks' gestations) were analyzed for MMP-2, MMP-9, TIMP-1, and TIMP-2. Data normalized by TA values of free secretory component of immunoglobulin A were compared for CLD (n = 22) versus no CLD (n = 27). Also, known clinical predictors of CLD (gestational age, birth weight, and sex) were assessed for both groups. Association of predictors with the outcome CLD was assessed by logistic regression. Mean gestational age was lower in CLD infants, but birth weight and gender were comparable for both groups. CLD infants had significantly lower TIMP-1 level with higher MMP-9/TIMP-1 ratio during the first 2 weeks of life and low TIMP-2 and MMP-2 levels during the first 3 days of life compared with no-CLD infants. Logistic regression analysis indicated that the findings are predictive of CLD. We conclude that low tracheal aspirate levels of TIMPs, with a high MMP-9/TIMP-1 ratio early in life, are associated with subsequent development of CLD.

Detection of microorganisms in the tracheal aspirates of preterm infants by polymerase chain reaction: association of adenovirus infection with bronchopulmonary dysplasia.

Bronchopulmonary dysplasia (BPD) is recognized as an important cause of morbidity and mortality in preterm infants. Because the role of congenital infections in BPD has been debated, the purpose of this study was to test the hypothesis that detection of infectious agents in tracheal aspirate samples was associated with the development of BPD. Tracheal aspirate samples were obtained within the 1st week of life and screened by polymerase chain reaction for adenovirus, cytomegalovirus, parvovirus, enteroviruses, Ureaplasma urealyticum, Mycoplasma hominis, Mycoplasma pneumoniae, and Chlamydia species. BPD was defined as persistent oxygen dependence at 28 d of age and 36 wk postconceptional age (PCA). Infants that expired before these time points were excluded from statistical analysis. Out of 89 infants studied, at 28 d of life, 13 had expired, 45 had BPD, and 31 had no BPD (controls). At 36 wk PCA, 15 infants expired, 39 still had BPD, and 35 did not. A significant increase in the frequency of adenovirus genome was identified in BPD patients compared with controls, both at 28 d of life (12/45 = 27% versus 1/31 = 3%: p< or =0.01) and at 36 wk PCA (10/39 = 29% versus 2/35 = 6%: p = 0.01). Other microorganisms were rarely detected and not associated with the development of BPD. This is the first study reporting the frequency of detection of adenovirus DNA in tracheal aspirate samples obtained during the 1st week of life from infants with BPD and suggests that prenatal acquisition may be important in the development of BPD.