RESUMEN
Division of large, immature alveolar structures into smaller, more numerous alveoli increases the surface area available for gas exchange. Alveolar division requires precise epithelial-mesenchymal interactions. However, few experimental models exist for studying how these cell-cell interactions produce changes in 3-dimensional structure. Here we report an epithelial-mesenchymal cell co-culture model where 3-dimensional peaks form with similar cellular orientation as alveolar structures in vivo. Co-culturing fetal mouse lung mesenchyme with A549 epithelial cells produced tall peaks of cells covered by epithelia with cores of mesenchymal cells. These structures did not form when using adult lung fibroblasts. Peak formation did not require localized areas of cell proliferation or apoptosis. Mesenchymal cells co-cultured with epithelia adopted an elongated cell morphology closely resembling myofibroblasts within alveolar septa in vivo. Because inflammation inhibits alveolar formation, we tested the effects of E. coli lipopolysaccharide on 3-dimensional peak formation. Confocal and time-lapse imaging demonstrated that lipopolysaccharide reduced mesenchymal cell migration, resulting in fewer, shorter peaks with mesenchymal cells present predominantly at the base. This epithelial-mesenchymal co-culture model may therefore prove useful in future studies of mechanisms regulating alveolar morphogenesis.
Asunto(s)
Células Epiteliales/fisiología , Transición Epitelial-Mesenquimal/fisiología , Mesodermo/citología , Mesodermo/fisiología , Morfogénesis/fisiología , Alveolos Pulmonares/citología , Alveolos Pulmonares/crecimiento & desarrollo , Animales , Comunicación Celular/fisiología , Movimiento Celular , Tamaño de la Célula , Células Cultivadas , Técnicas de Cocultivo/métodos , Células Epiteliales/citología , Ratones , Ratones Endogámicos BALB C , Modelos AnimalesRESUMEN
Bronchopulmonary dysplasia (BPD) is a frequent complication of preterm birth. This chronic lung disease results from arrested saccular airway development and is most common in infants exposed to inflammatory stimuli. In experimental models, inflammation inhibits expression of fibroblast growth factor-10 (FGF-10) and impairs epithelial-mesenchymal interactions during lung development; however, the mechanisms connecting inflammatory signaling with reduced growth factor expression are not yet understood. In this study we found that soluble inflammatory mediators present in tracheal fluid from preterm infants can prevent saccular airway branching. In addition, LPS treatment led to local production of mediators that inhibited airway branching and FGF-10 expression in LPS-resistant C.C3-Tlr4(Lpsd)/J fetal mouse lung explants. Both direct NF-κB activation and inflammatory cytokines (IL-1ß and TNF-α) that activate NF-κB reduced FGF-10 expression, whereas chemokines that signal via other inflammatory pathways had no effect. Mutational analysis of the FGF-10 promoter failed to identify genetic elements required for direct NF-κB-mediated FGF-10 inhibition. Instead, NF-κB activation appeared to interfere with the normal stimulation of FGF-10 expression by Sp1. Chromatin immunoprecipitation and nuclear coimmunoprecipitation studies demonstrated that the RelA subunit of NF-κB and Sp1 physically interact at the FGF-10 promoter. These findings indicate that inflammatory signaling through NF-κB disrupts the normal expression of FGF-10 in fetal lung mesenchyme by interfering with the transcriptional machinery critical for lung morphogenesis.
Asunto(s)
Factor 10 de Crecimiento de Fibroblastos/biosíntesis , Pulmón/embriología , FN-kappa B/metabolismo , Proteínas Quinasas/metabolismo , Animales , Corioamnionitis/metabolismo , Inmunoprecipitación de Cromatina , Femenino , Expresión Génica , Regulación de la Expresión Génica , Humanos , Inmunohistoquímica , Inmunoprecipitación , Recién Nacido , Pulmón/metabolismo , Ratones , Ratones Endogámicos BALB C , Embarazo , Nacimiento Prematuro , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de SeñalRESUMEN
The fetal lung vasculature forms in tandem with developing airways. Whereas saccular airway morphogenesis is arrested in bronchopulmonary dysplasia (BPD), the potential vascular phenotype in BPD at this stage of development is less well-understood. As inflammation increases the risk of BPD and induces arrest of saccular airway morphogenesis, we tested the effects of Escherichia coli LPS on fetal mouse lung vascular development. Injecting LPS into the amniotic fluid of Tie2-lacZ endothelial reporter mice at embryonic day 15 stimulated angiogenesis in the saccular stage fetal lung mesenchyme. LPS also increased the number of endothelial cells in saccular stage fetal mouse lung explants. Inflammation appeared to directly promote vascular development, as LPS stimulated pulmonary microvascular endothelial cell angiogenesis, cell migration, and proliferation in vitro. Whereas LPS did not increase expression of VEGF, angiopoietin-1 (Ang-1), Tie2, fetal liver kinase-1 (Flk-1), fms-like tyrosine kinase-1 (Flt-1), PDGFA, PDGFB, heparin-binding EGF-like growth factor (HB-EGF), or connective tissue growth factor (CTGF), LPS did stimulate the production of the angiogenic CC chemokines macrophage inflammatory protein-1α (MIP-1α) and monocyte chemoattractant protein-1 (MCP-1). Both MIP-1α and MCP-1 increased angiogenesis in fetal mouse lung explants. In addition, inhibitory antibodies against MIP-1α and MCP-1 blocked the effects of LPS on fetal lung vascular development, suggesting these chemokines are downstream mediators of LPS-induced angiogenesis. We speculate that an inflammation-mediated surge in angiogenesis could lead to formation of aberrant alveolar capillaries in the lungs of patients developing BPD.
Asunto(s)
Quimiocinas CC/metabolismo , Corioamnionitis/inmunología , Pulmón/irrigación sanguínea , Pulmón/embriología , Neovascularización Fisiológica , Animales , Displasia Broncopulmonar/embriología , Displasia Broncopulmonar/etiología , Displasia Broncopulmonar/inmunología , Estudios de Casos y Controles , Quimiocina CCL2/antagonistas & inhibidores , Quimiocina CCL2/farmacología , Quimiocina CCL3/antagonistas & inhibidores , Quimiocina CCL3/farmacología , Modelos Animales de Enfermedad , Células Endoteliales/efectos de los fármacos , Células Endoteliales/fisiología , Femenino , Humanos , Inmunidad Innata , Lactante , Recién Nacido , Lipopolisacáridos/farmacología , Pulmón/anomalías , Pulmón/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Neovascularización Fisiológica/efectos de los fármacos , Neovascularización Fisiológica/inmunología , EmbarazoRESUMEN
Mechanical ventilation is necessary and life saving in many neonates. Most complications are inherent to this intervention and cannot be confused with iatrogenic errors in judgment or care practices by clinicians. Clinical data suggest that complications such as volutrauma and air leak syndromes can negatively affect long-term pulmonary and non-pulmonary outcomes. Careful attention to many aspects of neonatal care, such as delivery room resuscitation, ventilatory support, and routine care practices, is needed to decrease pulmonary complications of mechanical ventilation. Clinical research is needed to improve mechanical ventilator strategies to reduce pulmonary complications and improve long-term outcomes.
Asunto(s)
Enfermedades Pulmonares/etiología , Respiración Artificial/efectos adversos , Barotrauma/etiología , Humanos , Enfermedad Iatrogénica , Recién Nacido , Intubación Intratraqueal/efectos adversos , Intubación Intratraqueal/instrumentación , Lesión Pulmonar , Atención Perinatal , Neumotórax/etiología , Enfisema Pulmonar/etiologíaRESUMEN
PURPOSE OF REVIEW: Bronchopulmonary dysplasia continues to be an important cause of morbidity in premature infants who require mechanical ventilation. Management strategies have historically focused on normalizing blood gases but new research suggests that a higher PCO2 level may be well tolerated in premature infants. There are physiologic rationale and recent experimental data to support the potential benefits of permissive hypercapnia. RECENT FINDINGS: Higher PCO2 levels may allow a reduction in ventilatory support which reduces the risk of lung injury in intubated patients. Targeting PCO2 levels above 45 mmHg has been tested in randomized controlled trials. These trials report that neonates managed with permissive hypercapnia have a shorter duration of mechanical ventilation and reduced severity of bronchopulmonary dysplasia without an increase in adverse events. SUMMARY: Permissive hypercapnia appears as a safe and effective management strategy to decrease morbidity from bronchopulmonary dysplasia in premature infants. Although the preliminary results are promising, further research is needed to determine whether this strategy improves pulmonary outcomes without adverse effects.