RESUMEN
Serotonin [5-hydroxytryptamine (5-HT)] biosynthesis depends on two rate-limiting tryptophan hydroxylases (Tph): Tph1, which is expressed in peripheral organs, and Tph2, which is expressed in neurons. Because 5-HT is involved in pulmonary hypertension (PH), we investigated whether genetic variations in Tph1 and/or Tph2 affected PH development in mice. To examine the functional impact of peripheral Tph1 deficiency on hypoxic PH, we used Tph1(-/-) mice characterized by very low 5-HT synthesis rates and contents in the gut and lung and increased 5-HT synthesis in the forebrain. With chronic hypoxia, 5-HT synthesis in the forebrain increased further. Hypoxic PH, right ventricular hypertrophy, and distal pulmonary artery muscularization were less severe (P < 0.001) than in wild-type controls. The Tph inhibitor p-chlorophenylalanine (100 mgxkg(-1)xday(-1)) further improved these parameters. We then investigated whether mouse strains harboring the C1473G polymorphism of the Tph2 gene showed different PH phenotypes during hypoxia. Forebrain Tph activity was greater and hypoxic PH was more severe in C57Bl/6 and 129X1/SvJ mice homozygous for the 1473C allele than in DBA/2 and BALB/cJ mice homozygous for the 1473G allele. p-Chlorophenylalanine reduced PH in all groups and abolished the difference in PH severity across mouse strains. Hypoxia increased 5-hydroxytryptophan accumulation but decreased 5-HT contents in the forebrain and lung, suggesting accelerated 5-HT turnover during hypoxia. These results provide evidence that dysregulation of 5-HT synthesis is closely linked to the hypoxic PH phenotype in mice and that Tph1 and Tph2 may contribute to PH development.
Asunto(s)
Hipertensión Pulmonar/etiología , Hipertensión Pulmonar/fisiopatología , Hipoxia/complicaciones , Polimorfismo Genético , Triptófano Hidroxilasa/deficiencia , Triptófano Hidroxilasa/genética , Animales , Enfermedad Crónica , Inhibidores Enzimáticos/farmacología , Fenclonina/farmacología , Hipertensión Pulmonar/genética , Mucosa Intestinal/metabolismo , Pulmón/metabolismo , Ratones , Ratones Endogámicos , Ratones Noqueados , Prosencéfalo/metabolismo , Arteria Pulmonar/fisiopatología , Serotonina/biosíntesis , Serotonina/metabolismo , Índice de Severidad de la EnfermedadRESUMEN
Lung cancer is the most frequent cause of cancer deaths. Its origin and development remain poorly understood, partly because of the lack of pertinent animal models. This study produced transgenic mice expressing the simian virus (SV) 40 T antigen (Tag) driven by a 1011 base-pair DNA fragment of the rat Calbindin-D9K (CaBP9K) promoter. All transgenic animals developed multifocal pulmonary tumours with pathological and ultrastructural features consistent with adenocarcinomas. Using immunohistochemistry, northern blot or western blot, tumours were found to express the transcription factor TTF-1, as well as specific markers of the peripheral airway Clara cells (CC10) and alveolar type II cells (surfactant proteins A, B, C, and D). This model, with its similarities to human adenocarcinoma, should be useful not only for addressing the mechanisms underlying the development and progression of lung cancer, but also for testing new therapeutic approaches.
Asunto(s)
Adenocarcinoma/genética , Antígenos Transformadores de Poliomavirus/genética , Neoplasias Pulmonares/genética , Regiones Promotoras Genéticas/genética , Proteína G de Unión al Calcio S100/genética , Uteroglobina , Animales , Northern Blotting , Western Blotting , Calbindinas , Cromogranina A , Cromograninas/metabolismo , Modelos Animales de Enfermedad , Ratones , Ratones Transgénicos , Microscopía Electrónica , Proteínas Nucleares/metabolismo , Proteínas/metabolismo , Surfactantes Pulmonares/metabolismo , Ratas , Factor Nuclear Tiroideo 1 , Factores de Transcripción/metabolismo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Congenital diaphragmatic hernia (CDH) is a major cause of refractory respiratory failure in the newborn. Besides pulmonary hypoplasia, the pathophysiology of CDH also includes surfactant deficiency. Vitamin A (vit A) is important for various aspects of lung development. We hypothesized that antenatal treatment with vit A would stimulate lung surfactant synthesis in experimental CDH induced in rats by maternal ingestion of the herbicide nitrofen (2,4-dichloro-phenyl-p-nitrophenyl-ether) on Day 12. Fetuses were assigned to six experimental groups: (1) controls from rats that received olive oil, the vehicle; (2) fetuses from rats that received olive oil on Day 12 and vit A orally (15,000 IU) on Day 14; (3) nitrofen (N)-exposed fetuses without diaphragmatic hernia (N/no DH); (4) N/no DH from rats given vit A on Day 14; (5 ) nitrofen-exposed fetuses with DH (N/+DH); (6) N/+DH from rats given vit A on Day 14. Fetuses were delivered by C-section at Day 21. Lung DNA content was lowered in the nitrofen group as compared with the controls group, but increased by subsequent vit A treatment. Lung surfactant disaturated phosphatidylcholine was reduced in the N/+DH group and restored to control level by vit A. The expression level of surfactant proteins (SP) -A and -C was decreased in vit A-treated control rats and in nitrofen-exposed fetuses with or without DH. Vit A restored SP-A and -C mRNA expression to control levels in N/+DH. SP-B expression was lowered in N/no DH and increased by vit A in this group. The proportion of type II cells assessed by SP-B immunolabeling was lowered in N/+DH and restored by vit A treatment. We conclude that antenatal treatment with vit A restores lung maturation in nitrofen-induced hypoplastic lungs with CDH. These findings point out vit A as a potential therapeutical agent for correcting surfactant deficiency in CDH.
Asunto(s)
Feto/efectos de los fármacos , Herbicidas/toxicidad , Hernia Diafragmática/prevención & control , Hernias Diafragmáticas Congénitas , Éteres Fenílicos/toxicidad , Surfactantes Pulmonares/biosíntesis , Vitamina A/farmacología , Análisis de Varianza , Animales , Northern Blotting , Cromatografía en Capa Delgada , ADN/análisis , Modelos Animales de Enfermedad , Femenino , Técnica del Anticuerpo Fluorescente , Edad Gestacional , Pulmón/citología , Pulmón/metabolismo , Masculino , Fosfatidilcolinas/análisis , Embarazo , Surfactantes Pulmonares/análisis , Surfactantes Pulmonares/deficiencia , Surfactantes Pulmonares/efectos de los fármacos , ARN Mensajero/análisis , Ratas , Ratas WistarRESUMEN
During late pregnancy, the fetal lung stores surfactant in preparation for extrauterine life. Surfactant deficiency, most often due to prematurity, precipitates respiratory distress syndrome (RDS) of the neonate. Although vitamin A (retinol) and retinoic acid have been shown to enhance the synthesis of phospholipid surfactant components, their effect on surfactant-specific proteins is unclear. No attempt has been made to evaluate the consequences of vitamin A restriction on surfactant phospholipid storage or on the expression of the life-essential surfactant protein-B (SP-B). We induced in rats a partial vitamin A deficiency leading to a 30-60% reduction in blood retinol, a status compatible with maintenance of gestation and absence of gross abnormalities in offspring. At term, lung surfactant phospholipids were reduced by 21%, and the major surfactant phospholipid, disaturated phosphatidylcholine (DSPC), was reduced by 27% in vitamin A-deficient (VAD) fetuses. The decrease in surfactant phospholipids and DSPC correlated linearly with plasma retinol, and reached about 50% in fetuses with the lowest retinol concentrations; it was accompanied by reduced expression of the gene for fatty acid synthase, a key enzyme in the synthetic pathway for surfactant-phospholipid lipid precursors. The amounts of SP-A, SP-B, and SP-C messenger RNAs were decreased by 46%, 32%, and 28%, respectively, in VAD fetuses. Consistently, amounts of SP-A and SP-B proteins were diminished as assessed by Western blotting. The proportion of type II cells determined after SP-B labeling was unchanged in VAD as compared with control lungs. Vitamin A deficiency is therefore a cause of lung maturational delay. In view of its rather large incidence in human populations, it may represent an increased risk for RDS and an aggravating factor for prematurity.
Asunto(s)
Pulmón/embriología , Complicaciones del Embarazo , Preñez , Deficiencia de Vitamina A/embriología , Animales , Citidililtransferasa de Colina-Fosfato/análisis , Diterpenos , Femenino , Pulmón/enzimología , Fosfolípidos/análisis , Embarazo , Surfactantes Pulmonares/análisis , Ratas , Ratas Sprague-Dawley , Ésteres de Retinilo , Vitamina A/análogos & derivados , Vitamina A/análisis , Vitamina A/sangreRESUMEN
Keratinocyte growth factor (KGF) or fibroblast growth factor (FGF)-7, a peptide produced by stromal cells and in particular by lung mesenchyme, has recently been shown to influence early lung morphogenesis and to be a mitogen for fetal and adult alveolar type II cells. Although contradictory findings have been reported regarding its effects on surfactant protein expression, its effects on surfactant phospholipids have not been studied. We investigated the effects of KGF on the synthesis of surfactant components by cultured fetal rat type II cells isolated during the late gestational period, when surfactant accumulates in preparation for extrauterine life. We show that KGF is a potent stimulus of surfactant phospholipid synthesis, particularly for the major component of surfactant, disaturated phosphatidylcholine (DSPC). KGF increased choline incorporation into DSPC in a dose-dependent manner up to 25 ng/ml (1.3 x 10(-9) M), and this effect was greater for surfactant than for nonsurfactant DSPC. KGF was several times more potent in this respect than acidic FGF at the same molar concentration. KGF, similar to epidermal growth factor, also stimulated acetate incorporation and increased the surfactant phospholipid and DSPC content of cultured cells twofold. These effects correlated with increased choline phosphate cytidylyltransferase activity and increased fatty acid synthase activity and gene expression. KGF also induced a dose-dependent stimulation of surfactant protein-A, -B, and -C gene expression, leading to a 2- to 3-fold increase in their messenger RNAs. KGF therefore stimulates the synthesis of all surfactant components in developing type II cells at the time of surfactant accumulation. Its secretion by lung fibroblasts may thus be an important factor in promoting the maturation of fetal lung epithelium and the synthesis of sufficient surfactant. The results suggest that KGF could provide a new therapeutic agent for the management of the immature or injured lung.
Asunto(s)
Células Epiteliales/efectos de los fármacos , Factores de Crecimiento de Fibroblastos , Sustancias de Crecimiento/farmacología , Alveolos Pulmonares/efectos de los fármacos , Surfactantes Pulmonares/biosíntesis , Animales , Células Cultivadas , Factor de Crecimiento Epidérmico/farmacología , Células Epiteliales/citología , Factor 10 de Crecimiento de Fibroblastos , Factor 7 de Crecimiento de Fibroblastos , Fibroblastos/metabolismo , Expresión Génica , Morfogénesis , Alveolos Pulmonares/citología , Alveolos Pulmonares/embriología , Surfactantes Pulmonares/genética , RatasRESUMEN
Human peritoneal dialysis effluent (PDE) contains a phosphatidylcholine-rich compound similar to the surfactant that lines lung alveoli. This material is secreted by mesothelial cells. Lung surfactant is also characterized by four proteins essential to its function. After having long been considered as lung-specific, some of them have been found in gastric and intestinal epithelial cells. To explore further the similarity between lung and peritoneal surfactants, we investigated whether mesothelial cells also produce surfactant proteins. We used rat transparent mesentery, human visceral peritoneum biopsies and PDE. Surfactant proteins were searched for after one- and two-dimensional SDS/PAGE and Western blotting. On a one-dimensional Western blot, bands at 38 and 66 kDa in rat mesentery, and at 38 and 66 kDa in human peritoneal mesothelial cells (in vivo and in vitro) and PDE, corresponded to monomeric and dimeric forms of lung surfactant protein A (SP-A). On two-dimensional Western blots, the 32 and 38 kDa spots in mesentery and PDE localized at the acidic pH appropriate to the SP-A monomer's isoelectric point. SP-D was also identified at the same 43 kDa molecular mass as in lung. SP-B was not detected in mesenteric samples. Expression of SP mRNA species was also assessed by reverse transcriptase-PCR, which was performed with specific primers of surfactant protein cDNA sequences. With primers of SP-A and SP-D, DNA fragments of the same size were amplified in lung and mesentery, indicating the presence of SP-A and SP-D mRNA species. These fragments were labelled by appropriate probes in a Southern blot. No amplification was obtained for SP-B. These results show that mesentery cells produce SP-A and SP-D, although they are of embryonic origin (mesodermal) and are different from those of the lung and digestive tract (endodermal) that secrete these surfactants.