Search

Exocytosis and endocytosis are the way of macromolecules transmembrane transport. Pulmonary surfactant (PS), one of such macromolecules, is secreted via exocytosis of lamellar bodies and recycled via endocytosis by type II alveolar epithelial cells (AEC II). It maintains low alveolar surface tension and is therefore essential to normal lung function. PS deficiency causes respiratory distress syndrome in infants. Congenital diaphragmatic hernia is an abnormal condition in which low lung compliance is involved. This condition is multifactorial and a primary surfactant deficiency may be responsible for it. We hypothesize that surfactant deficiency is involved in CDH and depressed activity of exocytosis and endocytosis in AEC II is responsible for the surfactant deficiency in the lung of newborn with congenital diaphragmatic hernia.

Background: Pulmonary hypoplasia and persistent pulmonary hypertension account for significant mortality and morbidity in neonates with congenital diaphragmatic hernia (CDH). Global lung immaturity and studies in animal models suggest the presence of surfactant deficiency that may further complicate the pathophysiology of CDH. However, data about surfactant status in human fetuses with CDH at birth are contradictory. The lack of a chronological study of surfactant content in late pregnancy has been a significant limitation. The appropriateness of administering surfactant supplements to neonates with CDH is therefore a debated question., Methods and Findings: We investigated surfactant content in human fetuses with CDH compared to age-matched fetuses with nonpulmonary diseases used as controls. Concentrations of disaturated phosphatidylcholine and surfactant proteins were found to be similar at a given stage of pregnancy, with both components showing a similar pattern of increase with progressing pregnancy in fetuses with CDH and in control fetuses. Thyroid transcription factor 1, a critical regulator of surfactant protein transcription, similarly displayed no difference in abundance. Finally, we examined the expression of three glucocorticoid-regulated diffusible mediators involved in lung epithelial maturation, namely: keratinocyte growth factor (KGF), leptin, and neuregulin 1 beta 1 (NRG1-beta1). KGF expression decreased slightly with time in control fetuses, but remained unchanged in fetuses with CDH. Leptin and NRG1-beta1 similarly increased in late pregnancy in control and CDH lungs. These maturation factors were also determined in the sheep fetus with surgical diaphragmatic hernia, in which surfactant deficiency has been reported previously. In contrast to the findings in humans, surgical diaphragmatic hernia in the sheep fetus was associated with decreased KGF and neuregulin expression. Fetoscopic endoluminal tracheal occlusion performed in the sheep model to correct lung hypoplasia increased leptin expression, partially restored KGF expression, and fully restored neuregulin expression., Conclusions: Our results indicate that CDH does not impair surfactant storage in human fetuses. CDH lungs exhibited no trend toward a decrease in contents, or a delay in developmental changes for any of the studied surfactant components and surfactant maturation factors. Surfactant amounts are likely to be appropriate to lung size. These findings therefore do not support the use of surfactant therapy for infants with CDH. Moreover, they raise the question of the relevance of CDH animal models to explore lung biochemical maturity.

In fetal sheep with surgically created diaphragmatic hernia (DH), tracheal occlusion (TO) can restore lung growth but does not ameliorate the increase in inter-alveolar wall thickness (T(W)). We determined whether prenatal exposure to glucocorticoids (GC) could reduce T(w) in fetuses with DH undergoing TO. At 65 days of gestation, DH was created in 12 fetal sheep, and TO subsequently performed at 110 days (DH/TO). Six of these fetuses were exposed to betamethasone (DH/TO + GC; 0.5 mg/kg; maternal, IM) 48 hr before delivery; Sham operated fetuses (n = 7) served as controls. At 139 days, we measured alveolar surface density (S(V)), parenchymal tissue fraction, T(W), alveolar type 2 (AE2) cell density and lung surfactant protein (SP) mRNA expression. Prenatal GC decreased T(W) and S(V) by 33% and 27% respectively, and increased fixed lung volume (by 55%), AE2 cell density and partially restored SPmRNA expression. Our data indicate that prenatal exposure to GC can reverse some of the negative effects of prolonged fetal TO. We hypothesize that a GC-induced reduction in lung liquid volume during TO contributes, in part, to the observed increase in AE2 cell density and SPmRNA expression.

Congenital diaphragmatic hernia (CDH) impairs fetal lung growth and increases the density of alveolar epithelial type 2 (AE2) cells. There is controversy whether surfactant protein (SP) expression is altered in CDH. The primary aim of this study was to assess SP expression (mRNA and protein) in the left and right lungs of fetal sheep with and without a diaphragmatic hernia (DH). Left-sided DH was created in four fetal sheep at 65 days of gestational age (g.a.). Sham-operated animals were used as controls. At 138 days g.a., lungs were harvested and the following parameters were measured: SP-A, -B, and -C mRNA expression (Northern blot), SP-A and -B expression (Western blot), and AE2 cell density (immunohistochemistry). The lung weight-to-body weight ratio was reduced by 42% in DH animals. The left-to-right lung weight ratio was lower in DH animals (0.47 +/- 0.03 vs. 0.69 +/- 0.03), indicative of asymmetric lung growth. SP-A, -B, and -C mRNA expression were increased by 61.7%, 32.9%, and 75.5%, respectively, in the left lungs of DH animals. SP-A and SP-B were also increased in DH. In the right lung, SP expression (mRNA and protein) was not different between groups. AE2 cell density was higher (by 67%) in the left but not right lungs of DH animals. Although DH in fetal sheep results in significant lung hypoplasia, SP expression is not reduced. On the contrary, SP expression was increased in the ipsilateral lung of fetuses with left-sided DH. Furthermore, AE2 cell density is increased in DH, suggesting that the increase in SP mRNA and protein levels is due to increases AE2 cell number. Our data further support the premise that fetal lung hypoplasia favors an AE2 phenotype., ((c) 2005 Wiley-Liss, Inc.)

With the use of stable isotope-labeled intravenous precursors for surfactant phosphatidylcholine (PC) synthesis, it has been shown that the de novo synthesis rates in preterm infants with respiratory distress syndrome (RDS) are very low as are turnover rates. This is consistent with animal data. Surfactant therapy does not inhibit endogenous surfactant synthesis, and prenatal corticosteroids stimulate it. With the use of stable isotope-labeled PC given endotracheally, surfactant pool size was estimated. It turned out to be low in RDS, as expected. Similar studies were performed in term neonates with severe lung diseases. In general, patients with lung injury show a lower surfactant synthesis. The controversy around surfactant in congenital diaphragmatic hernia (CDH) persists: studies on CDH with and without extracorporeal membrane oxygenation yielded different results. In severe meconium aspiration syndrome surfactant synthesis was found to be decreased but surfactant pool size was maintained. It is possible and safe to study surfactant metabolism in human neonates with the use of stable isotopes. This can help in answering clinical questions and has the potential to bring new in vitro and animal findings about surfactant metabolism to the patient., (Copyright 2005 S. Karger AG, Basel)

The aim of the study was to measure for the first time in humans surfactant disaturated-phosphatidylcholine (DSPC) net synthesis and kinetics by using a novel, dual stable isotope tracer approach. Ten infants with congenital diaphragmatic hernia [CDH; birth weight, 3.4 +/- 0.2; gestational age, 39.8 +/- 0.4 wk] and 6 age-matched control subjects with no lung disease (birth weight, 3.2 +/- 0.3 kg; gestational age, 39.1 +/- 1.1 wk), all of whom were admitted to the neonatal intensive care unit (Padua, Italy), were studied. All infants received simultaneously an intratracheal (carbon-13 di-palmitoyl-phosphatidylcholine) and an i.v. (deuterated palmitic acid) stable isotope tracer. Isotopic enrichment curves of DSPC from sequential tracheal aspirates were analyzed by mass spectrometry. DSPC kinetic data were expressed as mean +/- SEM and compared by the Mann-Whitney test. DSPC net synthesis from plasma palmitate was nearly identical in infants with CDH and control subjects (8.6 +/- 2.2 and 8.1 +/- 1.5 mg. kg(-1). d(-1); P = 0.7). DSPC apparent pool size was 36.7 +/- 7.5 and 58.5 +/- 9.1 mg/kg (P = 0.07) and half-life was 26.7 +/- 4.5 and 50.3 +/- 9.7 h (P = 0.03) in infants with CDH and control subjects, respectively. Both DSPC turnover and percentage of catabolism/recycling significantly correlated with duration of mechanical ventilation. In conclusion, the measurements of net DSPC synthesis and catabolism/recycling were reported for the first time in humans. Mean net DSPC synthesis was approximately 8 mg. kg(-1). d(-1). No significant differences were found between control subjects and infants with CDH. DSPC turnover was faster in infants with CDH, presumably reflecting an increased DSPC catabolism/recycling. Whether this may ultimately lead to a secondary surfactant deficiency in infants with CDH is still to be ascertained.

Background/purpose: The question of delayed lung maturation in congenital diaphragmatic hernia (CDH) is pending. Data about surfactant proteins (SPs) are sparse in human fetuses and discrepant in the ovine CDH model. The purpose of this study was to investigate, in the ovine surgically created CDH model, the expression of SPs and of thyroid transcription factor 1 (TTF-1), a key regulator of lung development that also controls the expression of surfactant proteins., Methods: Diaphragmatic hernia (DH) was created surgically in lamb fetuses on day 85 of gestation. On day 139, 5 DH and 6 control fetuses were retrieved by cesarean section. The mRNA levels for SPs and TTF-1 were determined by Northern blot analysis; SP-A and SP-B protein levels were assessed by Western blot analysis., Results: In DH lungs, SP-A, SP-B, and SP-C messenger RNAs were diminished by 82%, 67%, and 32%, respectively, compared with control level. SP-A and SP-B protein amounts were decreased consistently. TTF-1 expression was not altered in the surgical model., Conclusions: SP's deficiency appears to be a common feature of the various CDH models. By contrast with the nitrofen model, TTF-1 expression was not altered in the surgical model indicating different underlying molecular mechanisms in both models., (Copyright 2002, Elsevier Science (USA). All rights reserved.)

In animal models of congenital diaphragmatic hernia (CDH), surfactant deficiency contributes to the pathophysiology of the disease; however, information on CDH in humans is limited. We compared surfactant disaturated phosphatidylcholine (DSPC) synthesis and metabolism, by stable isotope technology, in newborn infants with CDH and in control subjects. DSPC amount, total proteins, and surfactant protein-A (SP-A) from tracheal aspirates were also measured. DSPC and SP-A were significantly lower in 14 infants with CDH than in the eight control subjects. Mean DSPC was 2.3 +/- 1.3 mg/ml of epithelial lining fluid (ELF) in infants with CDH and 4.6 +/- 1.5 mg/ml of ELF in control subjects (p = 0.001). Mean SP-A in infants with CDH and in control subjects was 16.2 +/- 9.3 and 61.2 +/- 30.6 microg/ml of ELF, respectively (p = 0.03). DSPC kinetics was measured in 12 of 14 infants with CDH and in 5 of 8 control subjects. Secretion time was 8.3 +/- 5.5 and 8.5 +/- 2.5 hours and peak time 51.9 +/- 15.2 and 51 +/- 13 hours in infants with CDH and in control subjects, respectively. Fractional synthesis rate was not different for infants with CDH and control subjects (p = 0.4). In conclusion, surfactant DSPC synthesis and kinetics were not significantly deranged in infants with CDH compared with control subjects. Other factors, such as lower surface area or increased DSPC catabolism, may contribute to surfactant pool alteration in CDH.

Background/purpose: Pulmonary hypoplasia is one of the main causes for the high mortality rate in patients with congenital diaphragmatic hernia (CDH). The expression of surfactant protein A in the hypoplastic CDH lung is reduced, and its concentration is decreased in the amniotic fluid of pregnancies complicated by CDH. In a CDH experimental model, prenatal glucocorticoid treatment has proved its efficacy in correcting the parameters of pulmonary biochemical and morphologic immaturity. The aim of this study was to investigate whether maternal administration of dexamethasone has any effect on the expression of surfactant protein A and surfactant protein B in nitrofen-induced experimental CDH rat model., Methods: CDH was induced in pregnant rats after administration of 100 mg of nitrofen on day 9.5 of gestation (term, 22 days). Dexamethasone (Dex, 0.25 mg/kg) was given by intraperitoneal injection on days 18.5 and 19.5 of gestation. Cesarean section was performed on day 21 of gestation. The fetuses were divided into 3 groups: group I, control (n = 16); group II, nitrofen-induced CDH (n = 16); group III, nitrofen-induced CDH with antenatal Dex treatment (n = 16). Indirect immunohistochemistry was performed using alkaline-phosphatase-coagulated streptavidin using anti-SP-A and anti-SP-B polyclonal antibodies. Reverse transcription polymerase chain reaction (RT-PCR) was performed to evaluate relative amount of SP-A and SP-B mRNA expression., Results: In the CDH lung (group II) we observed a markedly reduced number of type II pneumocytes positive for SP-A, and SP-B was increased to a level close to that of the control group. The relative amount of SP-A and SP-B was reduced significantly in group II compared with controls (P < .05) and significantly increased in group III compared with group II animals (P < .01)., Conclusion: These results suggest that antenatal glucocorticoid treatment increases the production of surfactant proteins in the CDH hypoplastic lung.

The hypoplastic lung in congenital diaphragmatic hernia (CDH) has both a quantitative and qualitative reduction in surfactant. Recently, the role of oxygen (O2) as a regulator of pulmonary surfactant-associated protein (SP) gene expression has been reported. The mRNA level of SP has been demonstrated to be increased in the lungs of animals exposed to hyperoxia. The aim of this study was to investigate SP mRNA expression in hypoplastic CDH lung in rats during mechanical ventilation in order to determine the effect of O2 on SP synthesis in CDH. A CDH model was induced in pregnant rats following administration of nitrofen. The newborn rats with CDH and controls were intubated and ventilated. Ventilation was continued for 6 h under 100% oxygen. Reverse-transcription polymerase chain reaction (RT-PCR) was performed to evaluate the relative amounts of mRNA expression of SP-A, SP-B, SP-C, and SP-D. Relative amounts of SP-A, SP-B, and SP-D mRNA expression in CDH lung were significantly decreased compared to controls at birth and 6 h after ventilation. There was no significant difference in SP-C mRNA expression between CDH animals and controls. Upregulated mRNA expression of SP-A, SP-B, and SP-D in lungs of control animals at 6 h after ventilation suggests that oxygenation accelerates postnatal SP synthesis in normal lungs. The inability of O2 to increase SP mRNA expression in hypoplastic CDH lung suggests that the hypoplastic lung is not responsive to increased oxygenation for the synthesis of SP.

Objectives: Infants with congenital diaphragmatic hernia may have biochemically immature lungs. However, normal lecithin/sphingomyelin ratios and phosphatidylglycerol concentrations have been reported in the amniotic fluid of congenital diaphragmatic hernia patients. We hypothesized that if the lungs of congenital diaphragmatic hernia patients are surfactant deficient, that this condition would be reflected in an altered surfactant composition in the bronchoalveolar lavage fluid compared with that composition in age-matched controls., Design: Prospective, controlled study., Setting: Surgical intensive care unit in a Level III pediatric university hospital., Patients: Four groups were studied: two groups of congenital diaphragmatic hernia patients (conventionally ventilated, n = 13; treated with extracorporeal membrane oxygenation, n = 5); and two control groups (conventionally ventilated, n = 13; extracorporeal membrane oxygenation, n = 6)., Interventions: Bronchoalveolar lavage, using a blind, standardized technique, was performed in conventionally ventilated congenital diaphragmatic hernia patients, extracorporeal membrane oxygenation-treated congenital diaphragmatic hernia patients, age-matched conventionally ventilated controls without pulmonary abnormalities, and extracorporeal membrane oxygenation-treated infants without congenital diaphragmatic hernia., Measurements and Main Results: The concentrations of different surfactant phospholipids and the fatty acid composition of phosphatidylcholine in bronchoalveolar lavage fluid were measured. No significant differences between the concentrations of phosphatidylcholine and phosphatidylglycerol, and the lecithin/sphingomyelin ratios, were found between the four groups. The fatty acid composition of phosphatidylcholine in conventionally ventilated patients showed a median percentage of palmitic acid within the normal range for age in both groups: 68% in congenital diaphragmatic hernia patients and 73% in controls (p < .001)., Conclusions: Our findings indicate that the concentrations of different phospholipids are similar in congenital diaphragmatic hernia patients and controls without congenital diaphragmatic hernia. A primary surfactant deficiency is unlikely in infants with congenital diaphragmatic hernia. However, secondary surfactant deficiency after respiratory failure may be involved.

We hypothesized that the expression of surfactant protein A (SP-A) would be altered in developing lungs from rat fetuses with congenital diaphragmatic hernia (CDH) induced by maternal ingestion of 2,4-dichlorophenyl-p-nitrophenyl ether (Nitrofen) on Day 9 of gestation. We compared our findings in fetuses exposed to Nitrofen with a CDH with those in Nitrofen-exposed fetuses without a CDH, and control fetuses whose mothers received olive oil only, the vehicle for Nitrofen. In late gestation, immunocytochemistry using a polyclonal rabbit antihuman SP-A antibody revealed decreased amounts of this protein in lungs from fetuses with CDH. Using immunoblotting, the relative amount of SP-A on Day 21 of gestation was also decreased in lung tissue from fetuses with CDH compared with the other groups. Abnormalities of mRNA for SP-A were observed in both groups of Nitrofen-exposed fetuses compared with control rats. These findings suggest that there is decreased expression of SP-A in rat fetuses with CDH secondary to Nitrofen exposure.

In addition to pulmonary hypoplasia and an altered pulmonary vascular bed, the abnormal lung development caused by congenital diaphragmatic hernia (CDH) is associated with an impairment in the pulmonary surfactant system. The aim of this study was to correlate fetal serum cortisol and triiodothyronine (T3) levels, known indicators of surfactant development, with maturation of the surfactant system in the lamb CDH model. Analysis of the products of bronchoalveolar lavage in CDH showed decreased phospholipid (P < .01) and increased protein concentration (P < .02) per gram of lung tissue when compared with controls, but cortisol and T3 concentrations did not differ significantly between the two groups. These results suggest that the surfactant deficiency in CDH is not related to an alteration in cortisol or T3 levels but may be directly related to the mechanical effects of the intrathoracic bowel.

Infants with congenital diaphragmatic hernia (CDH) on extracorporeal membrane oxygenation (ECMO) can have initial lung atelectasis which, in survivors, gradually improves over time. To test the hypothesis that these patients could benefit from surfactant therapy, infants with CDH (born at > 34 weeks' gestation) on ECMO received either four doses of modified bovine lung surfactant extract (beractant) (surfactant group, n = 9) or an equal volume of air (control group, n = 8). Tracheal aspirate surfactant protein-A (SP-A) concentrations were initially low, and then increased over time in both CDH groups (P = .0021); however, levels remained low when compared with those of infants on ECMO who had other diagnoses (P = .04). Lung compliance (CL), time to extubation, time on oxygen, and total no. of hospital days were not different between the two groups. Infants with CDH had persistently elevated right ventricular pressure (RVP) at cessation of bypass when compared with non-CDH infants on ECMO (RVP = 53.25 mm Hg +/- 19.52 in the CDH group, 32.90 +/- 10.63 in the non-CDH group; P = .0121). The findings suggest that the postnatal surfactant deficiency may be more persistent in CDH infants than in non-CDH infants on ECMO. However, CDH remains a multifactorial condition, with delayed improvement, because of persistence of pulmonary hypertension, difficulties with vascular remodeling, degree of lung hypoplasia, or compromised respiratory mechanics.

This paper examines the amounts of tensoactive phospholipids in the lung tissue of rat fetuses treated with Nitrofen (TOK) and in control animals. The herbicide led to congenital diaphragmatic hernia (CDH) in some fetuses and to pulmonary hypoplasia (PH) in all. The amounts of phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylinositol (PI) and phosphatidylethanolamine (PE) per gram of fresh lung tissue were significantly increased in comparison with the control animals, those of phosphatidylserine (PS) and sphingomyelin (SM) were also increased, but not significantly. Fetuses with HP alone had intermediate values. These findings are in agreement with our previous demonstration of an excess of type II pneumocytes in this model, and point to the existence of some trouble of the secretion or release of surfactant in it; although they do no clarify whether the amount of alveolar surfactant is in fact decreased.