Ana Remesal

Dexamethasone and Betamethasone for Prenatal Lung Maturation: Differences in Vascular Endothelial Growth Factor Expression and Alveolarization in Rats

Background: Fetal and postnatal lung development is regulated by glucocorticoids. The use of antenatal corticosteroids is reported to produce effects on vascular endothelial growth factor (VEGF), which plays a crucial role in pulmonary development. Objectives: The purpose of this study was to compare pulmonary VEGF expression in newborn rats that were exposed to antenatal betamethasone versus dexamethasone and to evaluate its impact on the alveolarization period of rats (0–14 days of life). Methods: Betamethasone, dexamethasone or equivalent saline solution (control group) was administered to pregnant rats on 20th and 21st days of gestation. Pulmonary VEGF mRNA, VEGF protein expression, and alveolarization changes were evaluated at birth and at 14 days of life. Results: Betamethasone and dexamethasone were observed to have different actions on VEGF expression with a correlation with alveolarization on both days of study. Antenatal dexamethasone decreased VEGF expression, betamethasone tended to produce the induction of the expression of VEGF, and moreover, betamethasone did not produce a decrease in alveolarization as seen in the animals that received dexamethasone. Conclusions: Our results support the notion that betamethasone could be a better choice than dexamethasone for antenatal lung maturation.

Host and environmental factors influencing respiratory secretion of pro-wheezing biomarkers in preterm children.

To cite this article: Matías V, San Feliciano L, Fernández JE, Lapeña S, Garrido E, Ardura J, Soga MJ, Aragón MP, Remesal A, Benito F, Andrés J, Centeno F, Marugán V, Bachiller R, Bermejo‐Martin JF. Host and environmental factors influencing respiratory secretion of pro‐wheezing biomarkers in preterm children. Pediatric Allergy Immunology 2012: 23: 441–447.

Effects of Antenatal Betamethasone and Dexamethasone on the Lung Expression of Vascular Endothelial Growth Factor and Alveolarization in Newborn Rats Exposed to Acute Hypoxia and Recovered in Normoxia or Hyperoxia

The use of antenatal corticosteroids is widespread and it is important to know their effect(s) on vascular endothelial growth factor (VEGF), which plays a crucial role in pulmonary development. The purpose of this study was to compare pulmonary VEGF expression in newborn rats that were exposed to antenatal betamethasone versus dexamethasone under normoxia, hypoxia and oxidative stress, and to evaluate its impact on alveolarization. Betamethasone, dexamethasone or equivalent saline solution (control group) was administered to pregnant rats. Newborn rats were randomized to room air, hypoxia followed by hyperoxia, or hypoxia followed by air. Pulmonary VEGF protein, VEGF mRNA, and alveolarization were evaluated at 4 days of life. Betamethasone and dexamethasone were observed to have different actions on VEGF expression with a correlation with alveolarization. Antenatal dexamethasone decreased VEGF expression, and dexamethasone and hyperoxia had an additive effect on the inhibition of VEGF with a reduction in alveolar development. Betamethasone appeared to have an effect on the induction of the expression of VEGF, and it seemed to inhibit the negative action of hyperoxia on VEGF. Moreover, betamethasone did not produce a decrease in alveolarization. Our results support the notion that betamethasone could be better than dexamethasone for antenatal lung maturation.

Breast feeding and early life immunomodulation

To the Editor, We have read with interest the article from Belderbos et al. (1) suggesting the ability of breastfeeding to down-modulate the secretion of TNF-a and IL-10 by white blood cells in term children during the first month of life. Also very recently, we have reported the existence of an inverse association between duration of breastfeeding and levels of IL-10 and RANTES secreted by the upper respiratory tract 1 year after birth in preterm children (2). A total of 77 children were recruited in our study. Gestational age (weeks) was (mean, SD) 31.9 (2.9). Sex composition was (women/men): 35/38. Weight at birth (kg) was 1.8 (0.5). Length at birth (cm) was 42.5 (4.2). Eight children had broncho-pulmonary dysplasia at birth. The most frequent accompanying condition was the presence of personal (n = 16) or familiar antecedents (n = 32) of atopic dermatitis. The majority of the children received breastfeeding (60%) for an average of 5.4 months after birth. Cytokines were measured using the 27-plex Bio-Rad assay (Hercules, CA, USA) on a Luminex platform (Austin, TX, USA), including IL-1 receptor antagonist (IL-1RA), IL-9, IL-15, Eotaxin, human fibroblast growth factor–basic (FGF-b): IFN-inducible protein 10 (IP-10), macrophage inflammatory protein 1a (MIP1a), platelet-derived growth factor (PDGF-BB), regulated upon activation, normal T cell expressed, and secreted protein (RANTES), vascular endothelial growth factor (VEGF), IL1b, IL-6, IL-8, IL-7, IL-17, granulocyte colony-stimulating factor (G-CSF), monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 1b (MIP-1b), IL-2, IL-4, IL-5, IL-10, IL-12, IL-13, granulocyte macrophage colonystimulating factor (GM-CSF), IFN-c and tumor necrosis factor alpha (TNF-a). Interestingly, duration of breastfeeding (in months) inversely correlated with levels in nasopharyngeal aspirates of IL-1b (Spearman correlation coefficient, p): ( 0.254, 0,035), G-CSF ( 0.236, 0.050), MIP-1b ( 0.250, 0.039), IL-10 ( 0.323, 0,007), and TNF-a ( 0.254, 0,035) (Fig. 1), and also with RANTES, IL-6, IL-17 at the level (p < 0.1). Belderbos et al. normalized cytokines by means of logarithmic transformation. Reproducing this approach, we now used logarithmic values for cytokines. Univariate linear regression analysis was performed again to evaluate the relationship between breastfeeding and cytokine levels 1 year after birth. Potential confounding variables were also evaluated in the univariate analysis: gestational age, sex, weight at birth, height at birth, [breastfeeding (duration in months)], bronchopulmonary dysplasia, atopic dermatitis, smoking habit at home, assistance to daycare, siblings at daycare or at school, number of people living at home, sibilances needing of treatment in the first year of life, [prophylaxis with PVZ (three or more doses)], symptoms of respiratory infection in the first year of life observed by the pediatrician, and necessity of hospitalization in the first year of life. Those variables yielding p values <0.2 in the univariate analysis were entered into a further multivariate analysis. After adjusting for potential confounding variables, duration of breastfeeding was inversely associated with levels of RANTES and IL-10 (Table 1) In turn, the variable (siblings at daycare or at school) was directly associated with IL-10 levels and the variable (prophylaxis with PVZ) was inversely associated with levels of this cytokine. Both RANTES and IL-10 are involved in allergy/asthma pathogenesis. In conclusion, these works are pioneer in providing biological evidence on the immuno-modulatory ability of breastfeeding in early life. This activity could be explained by the protective effect of breastfeeding against infections (3, 4). Alternatively, breastfeeding-induced protection might rely on tolerance induction to common environmental and dietary antigens because of antigen transfer across mammary epithelium or to the presence of factors in breast milk influencing neonatal immune system maturation, including immunoglobulins, oligosaccharides, and antimicrobial proteins/peptides (5). These results could explain thus the potential protective effect of breastfeeding against asthma/allergy.

402 Effect of Antenatal Corticosteroids in Activity and Expression of Secretory Phospholipase A2 and TNF Alfa in Lung of Newborn Rats

Introduction The sPLA2 plays an important role in the development of acute respiratory distress syndrome. It is regulated by many factors including steroids and TNFa. Antenatal corticosteroids are recommended for preventing respiratory distress syndrome in preterm infants. Recent studies suggest that betamethasone might be a better choice than dexamethasone. The aim of this study is evaluate differences between both antenatal corticosteroids in the regulation of sPLA2 and TNFa. Methods Dexamethasone, betamethasone or saline were administered intravenously to pregnant Wistar rats on the 20th and 21st days of gestation. We evaluated pulmonary sPLA2 and TNFa mRNA in newborn rats at birth by RT-PCR. We also evaluated sPLA2 activity by an ultrasensitive non-radioactive method on microplate and the TNFa protein expression by ELISA. Differences between the groups were determined by one way ANOVA (p<0.05). Results We observed a statistically significant decrease in the sPLA2 mRNA in the betamethasone (0.61) and dexamethasone (0.26) groups respect the control (1.05) group and a decrease in the sPLA2 activity in the betamethasone group (33.78) respect the control group (50.74). We observed a statistically significant decrease in the TNFa protein in the betamethasone group (472.61) respect the dexamethasone group (768.65). Conclusions Antenatal glucocorticoids inhibits the expression of sPLA2 through the reduction of TNFa in the lung of newborn rats. These potential beneficial effects are more evident in the group treated with antenatal betamethasone. Our studies also support the notion that betamethasone could be the drug of choice for treating pregnant women at risk of preterm delivery.

Effect of prenatal steroidal inhibition of sPLA2 in a rat model of preterm lung

INTRODUCTION Secretory phospholipase A2 regulates surfactant catabolism and inflammatory cascade. This enzyme is correlated with compliance, oxygenation and major outcomes in various forms of acute respiratory failure. Steroids inhibit secretory phospholipase A2 in cell culture and are widely used to boost surfactant production before preterm delivery. No data are available about the effect of antenatal steroids on secretory phospholipase A2 in the offspring: we aimed to study this effect in a rat model of preterm lung. MATERIAL AND METHODS Fifteen pregnant Wistar rats were randomized to receive betamethasone, dexamethasone or placebo at 20 and 21 days gestation. Newborn rats were supported for 8 h and then sacrificed: lung tissue was analysed for secretory phospholipase A2 expression and activity, inflammatory mediators and protein content. Lipidomics was analysed using liquid chromatography-mass spectrometry. RESULTS Secretory phospholipase A2 expression was significantly reduced by antenatal steroids (p < 0.001). Secretory phospholipase A2 activity, TNFα and lysophosphatidylethanolamine, a product of phospholipase reaction, were lowest in betamethasone-treated rats (p < 0.001). There was a strong correlation between secretory phospholipase A2 activity and lysophosphatidylethanolamine (r = 0.75; p = 0.001) and this remained significant after adjustment for total proteins or phospholipids. CONCLUSIONS Antenatal steroids decrease secretory phospholipase A2 in rat model of preterm lung.

Effects of Oxidative Stress and Antenatal Corticosteroids on the Pulmonary Expression of Vascular Endothelial Growth Factor (VEGF) and Alveolarization

The lung is the human organ most susceptible to oxidative damage. The transition from fetal, a low-oxygen environment, to FiO2: 0.21, induces a relative degree of oxidative stress for all newborns, but especially in the case of preterm neonates whose lung development has been interrupted. A supplementary supply of oxygen (hyperoxia) is often used to treat preterm newborns and these infants may not yet be prepared to protect their lungs from oxidative injury.

489 Inhaled Nitric Oxide After Oxidative Stress in Newborn Rats. Effects on Vegf Expression and Alveolarization

Background and aims: Exposure of newborn rats to hyperoxia decreases VEGF expression and impairs alveolarization. Much evidence indicates that NO plays a role in VEGF signaling and recent studies have shown that NO induces VEGF synthesis. The purpose of this study was to determine whether inhaled NO improves VEGF expression in the lung tissue and alveolarization after neonatal exposure to oxidative stress. Methods: Newborn rats were randomized to breathe room air (A), 2 hours A + NO (20 ppm), 2 hours hypoxia + 2 hours hyperoxia (HH), HH + NO, 2 hours hypoxia + air (HA) or HA + NO. We evaluated pulmonary VEGF in rats at 14 days of life by inmunohistochemistry using 1:100 dilution of rabbit anti-human VEGF (sc-152, Santa Cruz). We used a semiquantitative assessment of VEGF inmunostained, were assigned a relative value from 0 (minimal staining) to 4 (most intense inmunostaining). Quantitative morphometric assessment was done on coded slides with 400x magnification and a eye piece with a sample square grid pattern (model CPLW 1018, Zeiss Optical, Hannover Md) and was done following the mathematical model of Weibel. Differences between the groups were determined by one way ANOVA (p< 0.01). Results: The VEGF was significantly decreased in the lungs of rats recovered in hyperoxia, it was correlated with a lower degree of alveolarization. Inhaled NO treatment after hyperoxia neither increase lung VEGF expression nor alveolarization. Conclusion: The inhaled NO did not improve the changes observed in rat lungs after hyperoxia exposure.