RESUMEN
BACKGROUND: Premature birth and respiratory distress syndrome (RDS) are risk factors for disturbed lung development and bronchopulmonary dysplasia (BPD). The molecular mechanisms related to prematurity and BPD remain largely unknown. Epithelial expression of the transcription factor GATA-6 has been implicated in normal and abnormal murine lung development. OBJECTIVES: The possible involvement of GATA-6 in the normal development and in RDS and BPD was investigated in the human and baboon lung. METHODS: Immunohistochemistry was used to study the expression of GATA-6 and thyroid transcription factor 1 in lung specimens from different age groups of human and baboon fetuses and newborns with lung disease. Furthermore, the regulatory role of TGF-ß1 in GATA-6 expression was investigated in human pulmonary epithelial cell lines using RT-PCR. RESULTS: GATA-6 expression increased in the developing human airway epithelium along with advancing gestation, but diminished to negligible at birth. In RDS, GATA-6 expression was enhanced at 5-7 days after birth, and decreased thereafter. In BPD, the expression of GATA-6 in alveolar epithelial cells was low. These results were confirmed and extended using an established baboon model of prematurity. The in vitro experiments revealed that TGF-ß1 induces GATA-6 and thyroid transcription factor 1 expression in lung epithelial cells. CONCLUSIONS: Our results suggest that the expression of GATA-6 at the early stages of the preterm lung may be related to impaired postnatal alveolar development.
Asunto(s)
Proteínas de Unión al ADN/biosíntesis , Factor de Transcripción GATA6/biosíntesis , Recien Nacido Prematuro/fisiología , Alveolos Pulmonares/fisiopatología , Síndrome de Dificultad Respiratoria del Recién Nacido/genética , Animales , Proteínas de Unión al ADN/genética , Femenino , Feto , Factor de Transcripción GATA6/genética , Regulación del Desarrollo de la Expresión Génica , Humanos , Inmunohistoquímica , Técnicas In Vitro , Recién Nacido , Masculino , Papio , Alveolos Pulmonares/citología , Alveolos Pulmonares/metabolismo , ARN/química , ARN/genética , Síndrome de Dificultad Respiratoria del Recién Nacido/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de TranscripciónAsunto(s)
Aminoácidos Dicarboxílicos/farmacología , Cobalto/farmacología , Deferoxamina/farmacología , Factor 1 Inducible por Hipoxia/metabolismo , Animales , Femenino , Humanos , Pulmón/efectos de los fármacos , Pulmón/embriología , Ratones , Neovascularización Fisiológica/efectos de los fármacos , Embarazo , Nacimiento Prematuro/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular/genética , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Respiratory distress syndrome (RDS) secondary to preterm birth and surfactant deficiency is characterized by severe hypoxemia, lung injury, and impaired production of nitric oxide (NO) and vascular endothelial growth factor (VEGF). Since hypoxia-inducible factors (HIFs) mediate the effects of both NO and VEGF in part through regulation by prolyl-hydroxylase-containing domains (PHDs) in the presence of oxygen, we hypothesized that HIF-1alpha and -2alpha in the lung are decreased following severe RDS in preterm neonatal lambs. To test this hypothesis, fetal lambs were delivered at preterm gestation (115-day gestation, term = 145 days; n = 4) and mechanically ventilated for 4 h. Lambs developed respiratory failure characterized by severe hypoxemia despite treatment with mechanical ventilation with high inspired oxygen concentrations. Lung samples were compared with nonventilated control animals at preterm (115-day gestation; n = 3) and term gestation (142-day gestation; n = 3). We found that HIF-1alpha protein expression decreased (P < 0.05) and PHD-2 expression increased (P < 0.005) at birth in normal term animals before air breathing. Compared with age-matched controls, HIF-1alpha protein and HIF-2alpha protein expression decreased by 80% and 55%, respectively (P < 0.005 for each) in preterm lambs with RDS. Furthermore, VEGF mRNA was decreased by 40%, and PHD-2 protein expression doubled in RDS lambs. We conclude that pulmonary expression of HIF-1alpha, HIF-2alpha, and the downstream target of their regulation, VEGF mRNA, is impaired following RDS in neonatal lambs. We speculate that early disruption of HIF and VEGF expression after preterm birth and RDS may contribute to long-term abnormalities in lung growth, leading to bronchopulmonary dysplasia.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Síndrome de Dificultad Respiratoria/metabolismo , Síndrome de Dificultad Respiratoria/fisiopatología , Animales , Animales Recién Nacidos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Proteínas de Unión al ADN/metabolismo , Modelos Animales de Enfermedad , Femenino , Edad Gestacional , Proteínas de Homeodominio/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Proteínas Inmediatas-Precoces/metabolismo , Pulmón/embriología , Pulmón/fisiopatología , Óxido Nítrico Sintasa de Tipo III/metabolismo , Embarazo , Procolágeno-Prolina Dioxigenasa/metabolismo , ARN Mensajero/metabolismo , Respiración Artificial , Síndrome de Dificultad Respiratoria/terapia , Índice de Severidad de la Enfermedad , Ovinos , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
Bronchopulmonary dysplasia (BPD), a chronic lung disease affecting preterm neonates, is associated with significant childhood and adult health problems. Histopathologic features of BPD include impaired vascular and distal airway development. We previously showed that activation of hypoxia-inducible factors (HIFs) by inhibition of prolyl hydroxylase domain-containing proteins (PHDs) is feasible and that it stimulates vascular endothelial growth factor (VEGF)-dependent angiogenesis in vitro. We tested the hypothesis that enhancement of angiogenesis by activation of HIFs improves lung growth and function in prematurely born neonates in vivo. Preterm baboons (125 day+14 day pro re nata O2 model, corresponding to 27 human gestational weeks) were treated for 14 days with intravenous (i.v.) FG-4095, a PHD inhibitor. Notably, 77% of diminished total alveolar surface area in untreated controls was recovered by FG-4095 treatment. Functional significance of the structural changes was indicated by improved oxygenation and lung compliance in FG-4095-treated newborns. Surfactant proteins B and C and saturated phosphatidylcholine were unchanged. Incidence of spontaneous ductus arteriosus closure was increased, likely contributing to lower ratio of pulmonary to systemic blood flow in FG-4095 group. These findings indicate that HIF stimulation by PHD inhibition ameliorates pathological and physiological consequences of BPD.
Asunto(s)
Factor 1 Inducible por Hipoxia/metabolismo , Enfermedades Pulmonares/tratamiento farmacológico , Pulmón/crecimiento & desarrollo , Nacimiento Prematuro , Animales , Animales Recién Nacidos , Enfermedad Crónica , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/farmacología , Femenino , Factor 1 Inducible por Hipoxia/fisiología , Pulmón/patología , Pulmón/fisiopatología , Enfermedades Pulmonares/etiología , Masculino , Neovascularización Fisiológica/efectos de los fármacos , Papio , Pruebas de Función Respiratoria , Resultado del TratamientoRESUMEN
Development of lung microvasculature is critical for distal airway formation. Both processes are arrested in the lungs of preterm newborns with bronchopulmonary dysplasia (BPD), a chronic form of lung disease. We hypothesized that activation of hypoxia-inducible factors (HIFs) augments lung vascular development. Pulmonary angiogenic factors were assessed by quantitative real-time PCR, Western blot, and immunohistochemistry in preterm baboons (125 days+14 days pro re nata O2 model) treated for 14 days with intravenous FG-4095, an inhibitor of prolyl hydroxylase domain-containing proteins (PHDs) that initiates HIF degradation. HIF-1alpha, but not HIF-2alpha, mRNA and protein were increased (8- and 3-fold, respectively) in FG-4095-treated baboons relative to untreated controls. Expression of PHD-1, -2, and -3 was unchanged. Of note, mRNA and/or protein for platelet-endothelial cell adhesion molecule 1 (PECAM-1) and vascular endothelial growth factor (VEGF) were increased by FG-4095. Moreover, PECAM-1-expressing capillary endothelial cells detected by immunohistochemistry were augmented in FG-4095-treated baboons to levels comparable to those in fetal age-matched controls. Alveolar septal cell expression of Ki67, a proliferative marker, and VEGF were similar in untreated controls and FG-4095-treated neonates. These results indicate that HIF stimulation by PHD inhibition enhances lung angiogenesis in the primate model of BPD.
Asunto(s)
Capilares/metabolismo , Células Endoteliales/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/fisiología , Pulmón/embriología , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Western Blotting , Capilares/citología , Capilares/efectos de los fármacos , Células Cultivadas , Sistemas de Computación , Células Endoteliales/efectos de los fármacos , Feto/irrigación sanguínea , Feto/metabolismo , Inmunohistoquímica , Pulmón/irrigación sanguínea , Papio , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/genética , Reacción en Cadena de la Polimerasa , Procolágeno-Prolina Dioxigenasa/genética , Procolágeno-Prolina Dioxigenasa/farmacología , Estructura Terciaria de Proteína , Proteínas/antagonistas & inhibidores , Proteínas/genética , ARN Mensajero/metabolismo , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
Diminished vascular and alveolar development is characteristic of bronchopulmonary dysplasia (BPD). The low fetal O(2) tension promotes angiogenic responses during ontogenesis, while preterm birth interrupts normal lung growth. Most of the angiogenic responses are governed by hypoxia-inducible factors (HIFs), the expressions of which are unknown in the lungs of preterm primates. Lung tissue was harvested from fetal third-trimester baboons as well as from preterm baboons (67% or 75% of term gestation) treated with mechanical ventilation and either pro re nata (PRN) or 100% O(2). Both groups of preterm animals developed lung hypoplasia similar to human BPD. Expression of HIF-1alpha protein by Western blotting of nuclear extracts of fetal baboon samples differed from that of HIF-2alpha in that both were high at early third trimester, but at term, HIF-1alpha was absent, whereas HIF-2alpha remained unchanged. Moreover, the expression of prolyl hydroxylase domain-containing proteins 2 and 3 (PHD-2 and -3), which degrade HIFs, was increased following term birth. HIF-1alpha was diminished both in 125-day and 140-day BPD models, whereas HIF-2alpha was reduced only in the latter. Surprisingly, vascular endothelial growth factor (VEGF) was enhanced in preterm baboons with BPD as compared with age-matched fetal controls, and there was a negative correlation between HIF-1alpha and/or HIF-2alpha and VEGF in BPD. Moreover, VEGF receptors KDR and/or Flt-1 were decreased in BPD. Preterm birth also prevented the end-gestational increase in the expression of endothelial cell marker platelet-endothelial cell adhesion molecule 1. These results suggest that selective downregulation of HIFs in lungs of preterm neonates may contribute to the pathophysiology of BPD.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Factor 1 Inducible por Hipoxia/metabolismo , Pulmón/metabolismo , Nacimiento Prematuro , Factor A de Crecimiento Endotelial Vascular/metabolismo , Actinas/metabolismo , Animales , Western Blotting , Femenino , Modelos Animales , Terapia por Inhalación de Oxígeno , Papio , Embarazo , Procolágeno-Prolina Dioxigenasa/metabolismo , Respiración Artificial , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Preterm neonates with respiratory distress syndrome (RDS) often develop a chronic form of lung disease called bronchopulmonary dysplasia (BPD), characterized by decreased alveolar and vascular development. Ventilator treatment with supraphysiological O2 concentrations (hyperoxia) contribute to the development of BPD. Hyperoxia down-regulates and hypoxia up-regulates many angiogenic factors in the developing lung. We investigated whether angiogenic responses could be augmented through enhancement of hypoxia-inducible factors 1alpha and 2alpha (HIF-1alpha and -2alpha, respectively) via blockade of prolyl hydroxylase domain-containing proteins (HIF-PHDs) in human microvascular endothelial cells from developing and adult lung, in epithelial A549 cells, and in fetal baboon explants in relative or absolute hyperoxia. PHD inhibitor (FG-4095) and positive control dimethyloxaloylglycine (DMOG), selective and nonselective HIF-PHD inhibitors, respectively, enhanced HIF-1alpha and -2alpha, vascular endothelial growth factor (VEGF), and platelet-endothelial cell adhesion molecule 1 expression in vitro in 95% and 21% O2. Furthermore, VEGF receptor fms-like tyrosine kinase 1 (Flt-1) was elevated, whereas kinase insert domain-containing receptor/fetal liver kinase 1 (KDR) was diminished in endothelial, but not epithelial, cells. Intracellular Flt-1 and KDR locations were unchanged by PHD blockade. Like VEGF, FG-4095 and DMOG increased angiogenesis in vitro, both in 95% and 21% O2, an effect that could be blocked through either Flt-1 or KDR. Notably, FG-4095 was effective in stimulating HIFs and VEGF also in fetal baboon lung explants. FG-4095 or DMOG treatment appeared to stimulate the feedback loop promoting HIF degradation in that PHD-2 and/or -3, but not PHD-1, were enhanced. Through actions characterized above, FG-4095 could have desirable effects in enhancing lung growth in BPD.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Hiperoxia/metabolismo , Pulmón/metabolismo , Neovascularización Fisiológica/fisiología , Procolágeno-Prolina Dioxigenasa/antagonistas & inhibidores , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Aminoácidos Dicarboxílicos/farmacología , Análisis de Varianza , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Western Blotting , Células Endoteliales/metabolismo , Ensayo de Inmunoadsorción Enzimática , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia , Neovascularización Fisiológica/efectos de los fármacos , Oxígeno/metabolismo , Papio , Reacción en Cadena de la Polimerasa , Molécula 1 de Adhesión Celular Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Diminished alveolar and vascular development is characteristic of bronchopulmonary dysplasia (BPD) affecting many preterm newborns. Hypoxia promotes angiogenic responses in developing lung via, for example, vascular endothelial growth factor (VEGF). To determine if prolyl 4-hydroxylase (PHD) inhibition could augment hypoxia-inducible factors (HIFs) and expression of angiogenic proteins essential for lung development, HIF-1alpha and -2alpha proteins were assessed in human developing and adult lung microvascular endothelial cells and alveolar epithelial-like cells treated with either the HIF-PHD-selective inhibitor PHI-1 or the nonselective PHD inhibitors dimethyloxaloylglycine (DMOG) and deferoxamine (DFO). PHI-1 stimulated HIF-1alpha and -2alpha equally or more effectively than did DMOG or DFO, enhanced VEGF release, and elevated glucose consumption, whereas it was considerably less cytotoxic than DMOG or DFO. Moreover, VEGF receptor Flt-1 levels increased, whereas KDR/Flk-1 decreased. PHI-1 treatment also increased PHD-2, but not PHD-1 or -3, protein. These results provide proof of principle that HIF stimulation and modulation of HIF-regulated angiogenic proteins through PHI-1 treatment are feasible, effective, and nontoxic in human lung cells, suggesting the use of PHI-1 to enhance angiogenesis and lung growth in evolving BPD.
Asunto(s)
Pulmón/enzimología , Procolágeno-Prolina Dioxigenasa/antagonistas & inhibidores , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Células Cultivadas , Cinamatos/farmacología , Inhibidores Enzimáticos/farmacología , Células Epiteliales/metabolismo , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia , Pulmón/citologíaRESUMEN
Pulmonary antioxidants and their therapeutic implications have been extensively studied during past decades. The purpose of this review is to briefly summarize the key findings of these studies as well as to elaborate on some novel approaches with respect to potential preventive treatments for neonatal chronic lung disease bronchopulmonary dysplasia (BPD). Such new ideas include, for example, modification of transcription factors governing the hypoxic response pathways, important in angiogenesis, cell survival, and glycolytic responses. The fundamental strategy behind that approach is that fetal lung normally develops under hypoxic conditions and that this hypoxic, growth-favoring environment is interrupted by a premature birth. Importantly, during fetal lung development, alveolar development appears to be dependent on vascular development. Therefore, enhancement of signaling factors that occur during hypoxic fetal life ('continued fetal life ex utero'), including angiogenic responses, could potentially lead to improved lung growth and thereby alleviate the alveolar and vascular hypoplasia characteristic of BPD.
Asunto(s)
Antioxidantes/metabolismo , Antioxidantes/uso terapéutico , Displasia Broncopulmonar/tratamiento farmacológico , Displasia Broncopulmonar/metabolismo , Recien Nacido Prematuro , Nacimiento Prematuro , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Displasia Broncopulmonar/etiología , Hipoxia de la Célula/fisiología , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia , Recién Nacido , Pulmón/efectos de los fármacos , Pulmón/embriología , Pulmón/crecimiento & desarrollo , Nacimiento Prematuro/etiología , Transactivadores/fisiología , Factores de Transcripción/fisiologíaRESUMEN
Preterm neonates with respiratory distress are exposed not only to the relative hyperoxia ex utero, but also to life-saving mechanical ventilation with high inspired oxygen (O2) concentrations, which is considered a major risk factor for the development of bronchopulmonary dysplasia, also referred to as chronic lung disease of infancy. O2 toxicity is mediated through reactive oxygen species (ROS). ROS are constantly generated as byproducts of normal cellular metabolism, but their production is increased in various pathological states, and also upon exposure to exogenous oxidants, such as hyperoxia. Antioxidants, either enzymatic or nonenzymatic, protect the lung against the deleterious effects of ROS. Expression of various pulmonary antioxidants is developmentally regulated in many species so that the expression is increased toward term gestation, as if in anticipation of birth into an O2-rich extrauterine environment. Therefore, the lungs of prematurely born infants may be ill-adapted for protection against ROS. While premature birth interrupts normal lung development, the clinical condition necessitating the administration of high inhaled O2 concentrations may lead to permanent impairment of alveolar development. An understanding of the processes involved in lung growth, especially in alveolarization and vascularization, as well as in repair of injured lung tissue, may facilitate development of strategies to enhance these processes.
Asunto(s)
Antioxidantes/uso terapéutico , Displasia Broncopulmonar/fisiopatología , Pulmón/metabolismo , Síndrome de Dificultad Respiratoria del Recién Nacido/fisiopatología , Animales , Antioxidantes/metabolismo , Displasia Broncopulmonar/tratamiento farmacológico , Humanos , Recién Nacido , Recien Nacido Prematuro , Pulmón/efectos de los fármacos , Síndrome de Dificultad Respiratoria del Recién Nacido/tratamiento farmacológicoRESUMEN
Induction or overexpression of pulmonary manganese superoxide dismutase (MnSOD) has been shown to protect against oxygen (O2) toxicity. Genetic inactivation of MnSOD (Sod2) results in multiple organ failure and early neonatal death. However, lungs or O2-tolerance of Sod2 knockout mice have not been investigated. We evaluated survival, lung histopathology, and other pulmonary antioxidants (glutathione cycle) of homozygous (-/-) and heterozygous (+/-) Sod2 mutant mice compared with wild-type controls (Sod2+/+) following 48 h exposure to either room air or to O2. The ability of antioxidant N-acetylcysteine to compensate for the loss of MnSOD was explored. Mortality of Sod2-/- mice increased from 0% in room air to 18 and 83% in 50 and 80% O2, respectively. N-acetylcysteine did not alter mortality of Sod2-/- mice. Histopathological analysis revealed abnormalities in saccules of Sod2-/- mice exposed either to room air or to 50% O2 suggestive of delayed postnatal lung development. In 50% O2, activities of glutamate-cysteine ligase (GCL) (previously known as gamma-glutamylcysteine synthetase, gamma-GCS) and glutathione peroxidase increased in Sod2-/- (35 and 70%, respectively) and Sod2+/- (12 and 70%, respectively) mice, but glutathione levels remained unaltered. We conclude that MnSOD is required for normal O2 tolerance and that in the absence of MnSOD there is a compensatory increase in pulmonary glutathione-dependent antioxidant defense in hyperoxia.