Epithelial Vegfa Specifies a Distinct Endothelial Population in the Mouse Lung.

The lung microvasculature is essential for gas exchange and commonly considered homogeneous. We show that VEGFA from the epithelium is required for a distinct endothelial cell (EC) population in the mouse lung. Vegfa is predominantly expressed by alveolar type 1 (AT1) cells and locally required to specify a subset of ECs. Single-cell RNA sequencing (scRNA-seq) reveals that ∼15% of lung ECs are transcriptionally distinct-marked by Carbonic anhydrase 4 (Car4)-and arise from bulk ECs, as suggested by trajectory analysis. Car4 ECs have extensive cellular projections and are separated from AT1 cells by a limited basement membrane without intervening pericytes. Car4 ECs are specifically lost upon epithelial Vegfa deletion; without Car4 ECs, the alveolar space is aberrantly enlarged despite the normal appearance of myofibroblasts. Lung Car4 ECs and retina tip ECs have common and distinct features. These findings support a signaling role of AT1 cells and shed light on alveologenesis.

Neonatal Lung Disease Associated with TBX4 Mutations.

Variable lung disease was documented in 2 infants with heterozygous TBX4 mutations; their clinical presentations, pathology, and outcomes were distinct. These findings demonstrate that TBX4 gene mutations are associated with neonatal respiratory failure and highlight the wide spectrum of clinicopathological outcomes that have implications for patient diagnosis and management.

Lung disease and pulmonary hypertension in the premature infant.

The premature infant is born into the world unprepared to naturally thrive in a foreign environment. Lung development entails immense growth, structural remodeling and differentiation of specialized cells during the normal term perinatal and postnatal periods. Thus, the premature infant presents with a lung deficient for appropriate respiration. Disruption of lung development seen in bronchopulmonary dysplasia (BPD) and chronic lung disease (CLD) results in not only impaired airway growth but also a deficiency in the accompanying vasculature including the capillary system required for gas exchange. Deficient vascular area can lead to elevated pulmonary vascular resistance and the development of pulmonary hypertension (PH). Unlike PH seen in children and adults with pulmonary arterial hypertension (PAH), treatment with conventional pulmonary vasodilators can be limited in developmental lung disease-associated PH because there are fewer blood vessels to dilate. In this brief review, we highlight some of the knowledge on PH in the premature infant presented at the Proceedings of the 22nd Annual Update on Pediatric and Congenital Cardiovascular Disease.

Post-infectious bronchiolitis obliterans.

Bronchiolitis obliterans is a rare and severe chronic lung disease resulting from a lower respiratory tract lesion. It may occur after a bone marrow or lung transplantation, infectious diseases, or less frequently after inhaling toxic substances or after connective tissue diseases. Pathology, pathogenesis, and molecular biology, as well as the best treatment of bronchiolitis obliterans, remain the subject of ongoing research. This review discusses our current knowledge of different areas of bronchiolitis obliterans associated with infectious lesions.

Tracking morphological complexities of organ development in culture.

Organogenesis is one of the most striking process during development. During this period, organ primordia pass throughout several stages in which the level of organisation increases in complexity to achieve the final organ architecture. Organ culture, a method in which an isolated organ is explanted and maintained ex-vivo, is an excellent tool for following the morphological dynamics during development. While most of the work has been made in early stages of development, culturing organs in mid-late stages is needed to understand the achievement of the final organ anatomy in the new-born. Here, we investigated the possibility of following morphological changes of the mice heart, lung, kidney and intestine using a filter-grid culture method for 7 days starting at E14.5. We observed that the anatomy, histology and survival of the cultured organs were indicative of a continuity of the developmental processes: they survived and morphodifferentiated during 5-7 days in culture. The exception was the heart, which started to die after 4 days. Using a second approach, we demonstrated that heart tissue can be easily cultured in body slices, together with other tissues such as the lung, with a healthier differentiation and longer survival. The culture method used here, permits a high-resolution imaging to identify the dynamic of organ architecture ex-vivo using morphovideos. We also confirmed the suitability of this system to perform lineage tracing using a vital dye in branching organs. In summary, this work tested the feasibility of monitoring and recording the anatomical changes that establish the final organ structure of the heart, lung, kidney and intestine. Additionally, this strategy allows the morphological study of organ development including fate maps with a relative long-term survival up to the onset of differentiation. This work contributes to elucidating how organs are formed, promoting the understanding of congenital malformations and to design organ replacement therapies.

Pre- and postnatal exposure of mice to concentrated urban PM2.5 decreases the number of alveoli and leads to altered lung function at an early stage of life.

Gestational exposure to air pollution is associated with negative outcomes in newborns and children. In a previous study, we demonstrated a synergistic negative effect of pre- and postnatal exposure to PM2.5 on lung development in mice. However, the means by which air pollution affects development of the lung have not yet been identified. In this study, we exposed pregnant BALB/c mice and their offspring to concentrated urban PM2.5 (from São Paulo, Brazil; target dose 600 µg/m3 for 1 h daily). Exposure was started on embryonic day 5.5 (E5.5, time of placental implantation). Lung tissue of fetuses and offspring was submitted to stereological and transcriptomic analyses at E14.5 (pseudoglandular stage of lung development), E18.5 (saccular stage) and P40 (postnatal day 40, alveolarized lung). Additionally, lung function and cellularity of bronchoalveolar lavage (BAL) fluid were studied in offspring animals at P40. Compared to control animals that were exposed to filtered air throughout gestation and postnatal life, PM-exposed mice exhibited higher lung elastance and a lower alveolar number at P40 whilst the total lung volume and cellularity of BAL fluid were not affected. Glandular and saccular structures of fetal lungs were not altered upon gestational exposure; transcriptomic signatures, however, showed changes related to DNA damage and its regulation, inflammation and regulation of cell proliferation. A differential expression was validated at E14.5 for the candidates Sox8, Angptl4 and Gas1. Our data substantiate the in utero biomolecular effect of gestational exposure to air pollution and provide first-time stereological evidence that pre- and early life-postnatal exposure compromise lung development, leading to a reduced number of alveoli and an impairment of lung function in the adult mouse.

Ventilation causes pulmonary vascular dilation and modulates the NOS and VEGF pathway on newborn rats with CDH.

BACKGROUND/PURPOSE: Congenital diaphragmatic hernia (CDH) is a defect that presents high mortality because of pulmonary hypoplasia and hypertension. Mechanical ventilation changes signaling pathways, such as nitric oxide and VEGF in the pulmonary arterioles. We investigated the production of NOS2 and NOS3 and expression of VEGF and its receptors after ventilation in rat fetuses with CDH. METHODS: CDH was induced by Nitrofen. The fetuses were divided into 6 groups: 1) control (C); 2) control ventilated (CV); 3) exposed to nitrofen (N-); 4) exposed to nitrofen ventilated (N-V), 5) CDH and 6) CDH ventilated (CDHV). Fetuses were harvested and ventilated. We assessed body weight (BW), total lung weight (TLW), TLW/BW ratio, the median pulmonary arteriolar wall thickness (MWT). We analyzed the expression of NOS2, NOS3, VEGF and its receptors by immunohistochemistry and Western blotting. RESULTS: BW, TLW, and TLW/BW ratio were greater on C than on N- and CDH (p<0.05). The MWT was higher in CDH than in CDHV (p<0.001). CDHV showed increased expression of NOS3 (p<0.05) and VEGFR1 (p<0.05), but decreased expression of NOS2 (p<0.05) and VEGFR2 (p<0.001) compared to CDH. CONCLUSION: Ventilation caused pulmonary vasodilation and changed the expression of NOS and VEGF receptors.

VEGF receptor expression decreases during lung development in congenital diaphragmatic hernia induced by nitrofen

Changes in vascular endothelial growth factor (VEGF) in pulmonary vessels have been described in congenital diaphragmatic hernia (CDH) and may contribute to the development of pulmonary hypoplasia and hypertension; however, how the expression of VEGF receptors changes during fetal lung development in CDH is not understood. The aim of this study was to compare morphological evolution with expression of VEGF receptors, VEGFR1 (Flt-1) and VEGFR2 (Flk-1), in pseudoglandular, canalicular, and saccular stages of lung development in normal rat fetuses and in fetuses with CDH. Pregnant rats were divided into four groups (n=20 fetuses each) of four different gestational days (GD) 18.5, 19.5, 20.5, 21.5: external control (EC), exposed to olive oil (OO), exposed to 100 mg nitrofen, by gavage, without CDH (N-), and exposed to nitrofen with CDH (CDH) on GD 9.5 (term=22 days). The morphological variables studied were: body weight (BW), total lung weight (TLW), left lung weight, TLW/BW ratio, total lung volume, and left lung volume. The histometric variables studied were: left lung parenchymal area density and left lung parenchymal volume. VEGFR1 and VEGFR2 expression were determined by Western blotting. The data were analyzed using analysis of variance with the Tukey-Kramer post hoc test. CDH frequency was 37% (80/216). All the morphological and histometric variables were reduced in the N- and CDH groups compared with the controls, and reductions were more pronounced in the CDH group (P<0.05) and more evident on GD 20.5 and GD 21.5. Similar results were observed for VEGFR1 and VEGFR2 expression. We conclude that N- and CDH fetuses showed primary pulmonary hypoplasia, with a decrease in VEGFR1 and VEGFR2 expression.

Evaluación del índice Doppler tiempo de aceleración/tiempo de eyección sistólico de la arteria pulmonar como indicador de desarrollo pulmonar en fetos pretérmino

Objetivos: Evaluar del índice Doppler tiempo de aceleración/tiempo de eyección sistólico de la arteria pulmonar, como indicador de desarrollo pulmonar en fetos pretérmino. Diseño: Estudio prospectivo y descriptivo. Institución: Hospital III Honorio Delgado, Arequipa, Perú. Participantes: Gestantes pretérmino. Intervenciones: Desde julio de 2011 hasta agosto de 2012, a fetos de gestantes entre 25 y 36 semanas de gestación, que acudieron al hospital para evaluación ecográfica de rutina sin evidencia de patología, se realizó Doppler del tronco de la arteria pulmonar y se correlacionó el índice tiempo de aceleración/tiempo de eyección sistólico (índice TA/TE) con la edad gestacional. Principales medidas de resultados: Valor promedio del índice por grupos de edad gestacional y comparación de los mismos. Resultados: Se evaluó 93 gestantes pretérmino con edad materna promedio de 22,4 años y se encontró una correlación lineal rho de 0,67 en la medición del índice TA/TE de la arteria pulmonar, en relación con la edad gestacional. Los valores promedio del índice TA/TE fueron 0,228 y 0,258 para las gestaciones de 29-30 y 31-32 semanas, respectivamente, habiendo diferencia significativa (p<0,05). Los valores medio del índice TA/TE para las gestaciones de 33-34 y 35-36 semanas fueron 0,272 y 0,292, respectivamente, siendo estadísticamente diferentes (p<0,05). Conclusiones: El índice Doppler tiempo de aceleración/tiempo de eyección sistólico de la arteria pulmonar mostró correlación lineal con la edad gestacional y sufrió un cambio significativo coincidente e indicador del desarrollo alveolar pulmonar.

Objectives: To determine fetal pulmonary artery acceleration time/systolic ejection time Doppler index as marker of pulmonary development in preterm fetuses. Design: Prospective, descriptive study. Setting: Hospital III Honorio Delgado, Arequipa, Peru. Participants: Preterm pregnant women with no evidence of pathology. Interventions: Between July 2011 and August 2012, correlation of acceleration time/systolic ejection time pulmonary artery Doppler index (AT/ET index) with gestational age was performed in pregnant women 25 through 36 weeks of gestation attended in routine hospital ultrasound evaluation. Main outcome measures: Mean index value by age groups and comparison. Results: In 93 preterm pregnant women 22.4 year-old average there was rho linear correlation in pulmonary artery AT/ET index estimation in relation to gestational age. AT/ET index average values were 0.228 and 0.258 for respectively 29-30 and 31-32 weeks with significant difference (p<0.05). AT/ET index values for 33-34 and 35-36 weeks gestations were respectively 0.272 and 0.292, statistically different (p<0.05). Conclusions: Acceleration time/systolic ejection time Doppler index of the fetal pulmonary artery showed linear correlation with gestational age and had a significant change coincident and marker of pulmonary alveolar development.

Embriología del desarrollo de los bronquios y el parénquima pulmonar / Embryology of development of the bronchi and lung parenchyma

Las malformaciones congénitas de aparato respiratorio son un extenso número de patologías que oscilan en gravedad dependiendo del momento embriológico en que se dio el fallo y cuales mecanismos fueron afectados. En la revisión se profundiza en patologías congénitas como el secuestro broncopulmonar y la malformación adenomatoide quística...

Effect of postnatal malnutrition on hyperoxia-induced newborn lung development

Several factors are associated with bronchopulmonary dysplasia. Among them, hyperoxia and lung immaturity are considered to be fundamental; however, the effect of malnutrition is unknown. Our objective was to evaluate the effects of 7 days of postnatal malnutrition and hyperoxia on lung weight, volume, water content, and pulmonary morphometry of premature rabbits. After csection, 28-day-old New Zealand white rabbits were randomized into four groups: control diet and room air (CA, N = 17), control diet and ¡Ý95% O2 (CH, N = 17), malnutrition and room air (MA, N = 18), and malnutrition and ¡Ý95% O2 (MH, N = 18). Malnutrition was defined as a 30% reduction of all the nutrients provided in the control diet. Treatments were maintained for 7 days, after which histological and morphometric analyses were conducted. Lung slices were stained with hematoxylin-eosin, modified orcein-resorcin or picrosirius. The results of morphometric analysis indicated that postnatal malnutrition decreased lung weight (CA: 0.83 ¡À 0.19; CH: 0.96 ¡À 0.28; MA: 0.65 ¡À 0.17; MH: 0.79 ¡À 0.22 g) and water content, as well as the number of alveoli (CA: 12.43 ¡À 3.07; CH: 8.85 ¡À 1.46; MA: 7.33 ¡À 0.88; MH: 6.36 ¡À 1.53 x 10-3/mm) and elastic and collagen fibers. Hyperoxia reduced the number of alveoli and increased septal thickening and the mean linear intercept. The reduction of alveolar number, collagen and elastic fibers was intensified when malnutrition and hyperoxia were associated. These data suggest that dietary restriction enhances the magnitude of hyperoxia-induced alveolar growth arrest and lung parenchymal remodeling. It is interesting to consider the important influence of postnatal nutrition upon lung development and ronchopulmonary dysplasia.