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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 immunohisto-chemistry 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.

Background: Leukemia inhibitory factor (LIF) and interleukin-6 (IL-6) are members of the family of the glycoprotein 130 (gp130)-type cytokines. These cytokines share gp130 as a common signal transducer, which explains why they show some functional redundancy. Recently, it was demonstrated that IL-6 promotes fetal lung branching. Additionally, LIF has been implicated in developmental processes of some branching organs. Thus, in this study LIF expression pattern and its effects on fetal rat lung morphogenesis were assessed., Methodology/principal Findings: LIF and its subunit receptor LIFRα expression levels were evaluated by immunohistochemistry and western blot in fetal rat lungs of different gestational ages, ranging from 13.5 to 21.5 days post-conception. Throughout all gestational ages studied, LIF was constitutively expressed in pulmonary epithelium, whereas LIFRα was first mainly expressed in the mesenchyme, but after pseudoglandular stage it was also observed in epithelial cells. These results point to a LIF epithelium-mesenchyme cross-talk, which is known to be important for lung branching process. Regarding functional studies, fetal lung explants were cultured with increasing doses of LIF or LIF neutralizing antibodies during 4 days. MAPK, AKT, and STAT3 phosphorylation in the treated lung explants was analyzed. LIF supplementation significantly inhibited lung growth in spite of an increase in p44/42 phosphorylation. On the other hand, LIF inhibition significantly stimulated lung growth via p38 and Akt pathways., Conclusions/significance: The present study describes that LIF and its subunit receptor LIFRα are constitutively expressed during fetal lung development and that they have an inhibitory physiological role on fetal lung 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.