Maria João Baptista

Ghrelin expression in human and rat fetal lungs and the effect of ghrelin administration in nitrofen-induced congenital diaphragmatic hernia

Ghrelin is a strong physiologic growth hormone secretagogue that exhibits endocrine and non-endocrine actions. In this study, ghrelin expression in humans and rats was evaluated throughout development of normal and hypoplastic lungs associated with congenital diaphragmatic hernia (CDH). Additionally, the effect of antenatal treatment with ghrelin in the nitrofen-induced CDH rat model was tested. In normal lungs, ghrelin was expressed in the primitive epithelium at early stages of development and decreased in levels of expression with gestational age. In hypoplastic lungs ghrelin was overexpressed in both human and rat CDH fetuses when compared with controls. Exogenous administration of ghrelin to nitrofen-treated dams led to an attenuation of pulmonary hypoplasia of CDH pups. Furthermore, the growth hormone, secretagogue receptor (GHSR1a), could not be amplified from human or rat fetal lungs by RT-PCR. In conclusion, of all the lungs studied so far, the fetal lung is one of the first to express ghrelin during development and might be considered a new source of circulating fetal ghrelin. Overexpression of ghrelin in hypoplastic lungs and the effect of exogenous administration of ghrelin to nitrofen-treated dams strongly suggest a role for ghrelin in mechanisms involved in attenuation of fetal lung hypoplasia, most likely through a GHSR1a-independent pathway.

IL-6 Is Constitutively Expressed During Lung Morphogenesis and Enhances Fetal Lung Explant Branching

Previous studies have shown that chorioamnionitis, with increased IL-6, promotes fetal lung maturation and decreases the incidence of respiratory distress syndrome in premature neonates. However, the expression pattern and the effects of IL-6 on fetal lung growth mechanisms remain unknown. IL-6 expression was assessed by in situ hybridization and by real-time PCR between 14.5 and 21.5 d postconception. Normal and nitrofen-induced hypoplastic lung explants were cultured with increasing IL-6 doses or IL-6 neutralizing antibodies. Branching, cellular proliferation (Ki-67) and MAPK phosphorylation in fetal lung explants were analyzed. Pulmonary primitive epithelium expressed IL-6 constitutively throughout all gestational ages, displaying highest levels during earliest stages. In normal and hypoplastic lung explants, IL-6 neutralizing antibodies significantly reduced, whereas IL-6 supplementation induced a biphasic effect (lower doses increased, while the highest dose did not accomplish additional effect) on branching and cellular proliferation. IL-6 enhanced p38-MAPK phosphorylation without changing MEK1/2 and JNK pathways. The present study suggests a physiological role for IL-6 on pulmonary branching mechanisms most likely involving p38-MAPK intracellular signalling pathway.

Fetal heart development in the nitrofen-induced CDH rat model: The role of mechanical and nonmechanical factors

BACKGROUND/PURPOSE In congenital diaphragmatic hernia (CDH), it was recently shown that early and late gestational lung underdevelopment is caused by nonmechanical and mechanical factors, respectively. Heart underdevelopment, which might predict lung hypoplasia, is commonly attributed to mechanical factors. The authors analyzed whether nonmechanical and mechanical factors affect cardiac growth and correlations between lung and heart weights during gestation. METHODS Left-sided CDH was induced in pregnant Wistar rats by administration of nitrofen on E9.5. At selected gestational ages (E18, E20, and E22), the lungs and heart were harvested, weighed, and analyzed for DNA and protein contents. Left lung and heart weights were correlated at those gestational ages. Two experimental groups: nitrofen without CDH (nitrofen), and nitrofen with CDH (CDH), were compared with normal controls. RESULTS At E18, both nitrofen-exposed groups presented similar and significant left lung (LL) hypoplasia. As gestation progressed (E20 and E22), in the nitrofen group left lung (LL) hypoplasia decreased, whereas in the CDH group LL hypoplasia was exacerbated relative to normal controls. In contrast, at E18 and E20, heart-to-body weight ratios as well as cardiac DNA and protein contents were reduced significantly in all animals exposed to nitrofen, with no significant differences observed between nitrofen and CDH groups. As gestation progressed, the difference between cardiac parameters in nitrofen-exposed and normal control rats diminished, and at E22 no significant differences were documented. In the CDH group, significant correlations were seen between lung and heart weights at E18 (r = 0.65; P <.05) and E20 (r = 0.4; P <.05), whereas at term gestation (E22) no significant correlation was observed (r = 0.21, not significant). CONCLUSIONS Nonmechanical factors, which might be directed by nitrofen, play a role in the pathogenesis of lung and heart hypoplasia manifested precociously in fetal life, whereas mechanical compression might influence only lung growth during late gestation. Heart weight predicts lung weight only in early gestational ages.

Local Fetal Lung Renin-Angiotensin System as a Target to Treat Congenital Diaphragmatic Hernia

Antenatal stimulation of lung growth is a reasonable approach to treat congenital diaphragmatic hernia (CDH), a disease characterized by pulmonary hypoplasia and hypertension. Several evidences from the literature demonstrated a possible involvement of renin-angiotensin system (RAS) during fetal lung development. Thus, the expression pattern of renin, angiotensin-converting enzyme, angiotensinogen, type 1 (AT1) and type 2 (AT2) receptors of angiotensin II (ANGII) was assessed by immunohistochemistry throughout gestation, whereas the function of RAS in the fetal lung was evaluated using fetal rat lung explants. These were morphometrically analyzed and intracellular pathway alterations assessed by Western blot. In nitrofen-induced CDH model, pregnant rats were treated with saline or PD-123319. In pups, lung growth, protein/DNA ratio, radial saccular count, epithelial differentiation and lung maturation, vascular morphometry, right ventricular hypertrophy and overload molecular markers, gasometry and survival time were evaluated. Results demonstrated that all RAS components were constitutively expressed in the lung during gestation and that ANGII had a stimulatory effect on lung branching, mediated by AT1 receptor, through p44/42 and Akt phosphorylation. This stimulatory effect on lung growth was mimicked by AT2-antagonist (PD-123319) treatment. In vivo antenatal PD-123319 treatment increased lung growth, ameliorated indirect parameters of pulmonary hypertension, improved lung function and survival time in nonventilated CDH pups, without maternal or fetal deleterious effects. Therefore, this study demonstrated a local and physiologically active RAS during lung morphogenesis. Moreover, selective inhibition of AT2 receptor is presented as a putative antenatal therapy for CDH.

N-terminal-pro-B type natriuretic peptide as a useful tool to evaluate pulmonary hypertension and cardiac function in CDH infants.

Objective: In congenital diaphragmatic hernia (CDH) the severity of pulmonary hypertension (PH) is considered, by several authors, determinant of clinical outcome. Plasmatic N-terminal-pro-B type natriuretic peptide (NT-proBNP) might be useful in diagnosis and management of PH in newborns, although its interest in CDH infants remains to be defined. Early NT-proBNP levels were assessed in CDH infants and correlated with cardiovascular echocardiographic parameters. Patients and Methods: 28 newborns, CDH and age-matched controls were enrolled in a prospective study. Clinical condition, NT-proBNP plasmatic levels, echo parameters of PH and biventricular function were assessed at 24 h after delivery as well as survival outcome. Results: Estimated mean pulmonary pressure and NT-proBNP were significantly higher in CDH than control infants. NT-proBNP significantly correlated with estimated pulmonary artery pressure, right ventricular Tei index, and tricuspid E/A ratio. Additionally, we found that CDH infants with NT-proBNP >11,500 pg/ml experienced a worse prognosis. Conclusions: We demonstrated that PH is associated with NT-proBNP elevation and diastolic impairment in CDH infants. Early elevations in NT-proBNP levels seem to alert for a subset of CDH infants with worse prognosis.

Paediatric dilated cardiomyopathy: clinical profile and outcome. The experience of a tertiary centre for paediatric cardiology.

Dilated cardiomyopathy is the most common form of cardiomyopathy in the paediatric population and an important cause of heart transplantation in children. The clinical profile and course of dilated cardiomyopathy in children have been poorly characterised. A retrospective review of 61 patients (37 female; 24 male) diagnosed with dilated cardiomyopathy from January, 2005 to June, 2012 at a single institution was performed. The median age at diagnosis was 15 months. Heart failure was present in 83.6% of patients and 44.3% required intensive care. The most prevalent causes were idiopathic (47.5%), viral myocarditis (18.0%) and inherited metabolic diseases (11.5%). In viral myocarditis, Parvovirus B19 was the most common identified agent, in concurrence with the increasing incidence documented recently. Inherited metabolic diseases were responsible for 11.5% of dilated cardiomyopathy cases compared with the 4-6% described in the literature, which reinforces the importance of considering this aetiology in differential diagnosis of paediatric dilated cardiomyopathy. The overall mortality rate was 16.1% and five patients underwent heart transplantation. In our series, age at diagnosis and aetiology were the most important prognosis factors. We report no mortality in the five patients who underwent heart transplantation, after 2 years of follow-up.

Persistent pulmonary hypertension of non cardiac cause in a neonatal intensive care unit.

Parenchymal lung diseases are the main cause of persistent pulmonary hypertension of the newborn (PPHN). We aimed to assess the non cardiac conditions associated to PPHN in the newborn and the survival rate over the last 15 years, at our center. A retrospective chart review of the neonates admitted for PPHN from 1996 to 2010 was performed. New therapies were introduced in 2003, and the survival rates between two periods (1996–2002 and 2003–2010) were compared. Out of 6750 newborns, 78 (1.1%) had the diagnosis of PPHN of non cardiac cause. The most prevalent causes were associated to pulmonary hypoplasia (30.7%), infection (24.3%), and aspiration syndromes (15.3%). Many other causes were identified in 33.3%. The overall survival rate was 68%. There was a significant difference on survival rates between the two periods (1996–2002 = 63.8% and 2003–2010 = 71.4%, P = 0.04). Our study showed a myriad of non cardiac aetiologies for PPHN of the newborn, most of them related to lung disease or lung hypoplasia. We observed an improvement in survival rate since 2003, which was associated to the use of new therapies.

Perinatal profile of ventricular overload markers in congenital diaphragmatic hernia

BACKGROUND In congenital diaphragmatic hernia (CDH), pulmonary hypertension increases right ventricle (RV) afterload, which could impair heart function and contribute to poor outcome for most affected infants. Nevertheless, the real significance of vascular pulmonary alterations in perinatal hemodynamics is largely unknown. It is defined that ventricular pressure overload induces increased myocardium gene expression of B-type natriuretic peptide (BNP) and components of the renin-angiotensinogen and endothelin (ET)-1 systems. Our aim was to evaluate perinatal myocardium expression of these genes associated with ventricular pressure overload in a nitrofen-induced CDH rat model. METHODS In the nitrofen-induced CDH rat model, fetuses from dated pregnant Sprague-Dawley rats at 15.5, 17.5, 19.5 and 21.5 days postcoitum as well as newborn pups were assigned to 3 experimental groups: control, nitrofen (exposed to nitrofen, without CDH), and CDH (exposed to nitrofen, with CDH). Myocardial samples collected from the RV and left ventricle (LV) were processed for quantification of messenger RNA (mRNA) of BNP, angiotensinogen, and ET-1. RESULTS The perinatal expression of BNP, angiotensinogen, and ET-1 mRNA in the RV and LV of the control group revealed daily changes. During gestation, the expression of BNP and angiotensinogen mRNA underwent significant oscillation compared with control in both nitrofen-exposed fetuses, although we cannot identify significant differences between the nitrofen and CDH groups. After birth, we found a significant increasing expression of all studied genes only in the RV of CDH pups. CONCLUSIONS Perinatal myocardial quantification of BNP, angiotensinogen, and ET-1 mRNA levels suggests that both nitrofen-exposed and control pups revealed prenatal variations of expression of the studied genes. Moreover, CDH is associated with significant molecular alterations only in the RV after birth.

A Novel Mutation in FOXF1 Gene Associated with Alveolar Capillary Dysplasia with Misalignment of Pulmonary Veins, Intestinal Malrotation and Annular Pancreas

Alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV) is a rare, fatal, neonatal developmental lung disorder, which usually presents as persistent pulmonary hypertension unresponsive to treatment. The authors report the case of a neonate with persistent pulmonary hypertension, associated with duodenal stenosis secondary to annular pancreas and intestinal malrotation. Support treatment, inhaled nitric oxide, oral sildenafil and nebulized iloprost were used with no clinical improvement. The neonate presented an overwhelming course, with hypoxemia refractory to treatment. At autopsy lung histology showed the characteristic features of ACD/MPV. DNA sequence analysis revealed a heterozygous nonsense mutation c.539C>A;p.S180X, in the first exon of FOXF1. FOXF1 has been identified as one of the genes responsible for ACD/MPV associated with multiple congenital malformations. This clinical case is the first report of a heterozygous nonsense mutation c.539C>A;p.S180X in the first exon of FOXF1, in a patient with ACD/MPV associated with annular pancreas and intestinal malrotation.

Brain-type natriuretic peptide in the diagnosis and management of persistent pulmonary hypertension of the newborn.

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