Sana Mujahid

miRNA regulated pathways in late stage murine lung development

BackgroundMicroRNAs play important roles in regulating biological processes, including organ morphogenesis and maturation. However, little is known about specific pathways regulated by miRNA during lung development. Between the canalicular and saccular stages of the developing lung several important cellular events occur, including the onset of surfactant synthesis, microvascular remodeling and structural preparation for subsequent alveolarization. The miRNAs that are actively regulated, and the identity of their targets during this important developmental interval in the lung remain elusive.ResultsUsing TLDA low density real-time PCR arrays, the expression of 376 miRNAs in male and female fetal mouse lungs of gestational days E15 – E18 were profiled. Statistical analyses identified 25 and 37 miRNAs that changed significantly between sexes and with gestation, respectively. In silico analysis using Ingenuity Pathway Analysis (IPA) identified specific pathways and networks known to be targets of these miRNAs which are important to lung development. Pathways that are targeted by sex regulated miRNAs include retinoin, IGFR1, Tp53 and Akt. Pathways targeted by gestation-regulated miRNAs include VEGFA and mediators of glucose metabolism.ConclusionMiRNAs are differentially regulated across time and between sexes during the canalicular and saccular stages of lung development. Sex-associated differential miRNA expression may regulate the differences in structural and functional male and female lung development, as shown by networks generated using in silico analysis. These data provide a valuable resource to further enhance the understanding of miRNA control of lung development and maturation.

MiR-221 and miR-130a regulate lung airway and vascular development.

Epithelial-mesenchymal interactions play a crucial role in branching morphogenesis, but very little is known about how endothelial cells contribute to this process. Here, we examined how anti-angiogenic miR-221 and pro-angiogenic miR-130a affect airway and vascular development in the fetal lungs. Lung-specific effects of miR-130a and miR-221 were studied in mouse E14 whole lungs cultured for 48 hours with anti-miRs or mimics to miR-130a and miR-221. Anti-miR 221 treated lungs had more distal branch generations with increased Hoxb5 and VEGFR2 around airways. Conversely, mimic 221 treated lungs had reduced airway branching, dilated airway tips and decreased Hoxb5 and VEGFR2 in mesenchyme. Anti-miR 130a treatment led to reduced airway branching with increased Hoxa5 and decreased VEGFR2 in the mesenchyme. Conversely, mimic 130a treated lungs had numerous finely arborized branches extending into central lung regions with diffusely localized Hoxa5 and increased VEGFR2 in the mesenchyme. Vascular morphology was analyzed by GSL-B4 (endothelial cell-specific lectin) immunofluorescence. Observed changes in airway morphology following miR-221 inhibition and miR-130a enhancement were mirrored by changes in vascular plexus formation around the terminal airways. Mouse fetal lung endothelial cells (MFLM-91U) were used to study microvascular cell behavior. Mimic 221 treatment resulted in reduced tube formation and cell migration, where as the reverse was observed with mimic 130a treatment. From these data, we conclude that miR-221 and miR-130a have opposing effects on airway and vascular morphogenesis of the developing lung.

Presenilin-1 Processing of ErbB4 in Fetal Type II Cells is Necessary for Control of Fetal Lung Maturation

Maturation of pulmonary fetal type II cells to initiate adequate surfactant production is crucial for postnatal respiratory function. Little is known about specific mechanisms of signal transduction controlling type II cell maturation. The ErbB4 receptor and its ligand neuregulin (NRG) are critical for lung development. ErbB4 is cleaved at the cell membrane by the γ-secretase enzyme complex whose active component is either presenilin-1 (PSEN-1) or presenilin-2. ErbB4 cleavage releases the 80kDa intracellular domain (4ICD), which associates with chaperone proteins such as YAP (Yes-associated protein) and translocates to the nucleus to regulate gene expression. We hypothesized that PSEN-1 and YAP have a development-specific expression in fetal type II cells and are important for ErbB4 signaling in surfactant production. In primary fetal mouse E16, E17, and E18 type II cells, PSEN-1 and YAP expression increased at E17 and E18 over E16. Subcellular fractionation showed a strong cytosolic and a weaker membrane location of both PSEN-1 and YAP. This was enhanced by NRG stimulation. Co-immunoprecipitations showed ErbB4 associated separately with PSEN-1 and with YAP. Their association, phosphorylation, and co-localization were induced by NRG. Confocal immunofluorescence and nuclear fractionation confirmed these associations in a time-dependent manner after NRG stimulation. Primary ErbB4-deleted E17 type II cells were transfected with a mutant ErbB4 lacking the γ-secretase binding site. When compared to transfection with wild-type ErbB4, the stimulatory effect of NRG on surfactant protein mRNA expression was lost. We conclude that PSEN-1 and YAP have crucial roles in ErbB4 signal transduction during type II cell maturation.

Pigment Epithelium–Derived Factor Mediates Impaired Lung Vascular Development in Neonatal Hyperoxia

Bronchopulmonary dysplasia is a chronic lung disease of preterm infants characterized by arrested microvascularization and alveolarization. Studies show the importance of proangiogenic factors for alveolarization, but the importance of antiangiogenic factors is unknown. We proposed that hyperoxia increases the potent angiostatin, pigment epithelium-derived factor (PEDF), in neonatal lungs, inhibiting alveolarization and microvascularization. Wild-type (WT) and PEDF(-/-) mice were exposed to room air (RA) or 0.9 fraction of inspired oxygen from Postnatal Day 5 to 13. PEDF protein was increased in hyperoxic lungs compared with RA-exposed lungs (P < 0.05). In situ hybridization and immunofluorescence identified PEDF production primarily in alveolar epithelium. Hyperoxia reduced alveolarization in WT mice (P < 0.05) but not in PEDF(-/-) mice. WT hyperoxic mice had fewer platelet endothelial cell adhesion molecule (PECAM)-positive cells per alveolus (1.4 ± 0.4) than RA-exposed mice (4.3 ± 0.3; P < 0.05); this reduction was absent in hyperoxic PEDF(-/-) mice. The interactive regulation of lung microvascularization by vascular endothelial growth factor and PEDF was studied in vitro using MFLM-91U cells, a fetal mouse lung endothelial cell line. Vascular endothelial growth factor stimulation of proliferation, migration, and capillary tube formation was inhibited by PEDF. MFLM-91U cells exposed to conditioned medium (CM) from E17 fetal mouse lung type II (T2) cells cultured in 0.9 fraction of inspired oxygen formed fewer capillary tubes than CM from T2 cells cultured in RA (hyperoxia CM, 51 ± 10% of RA CM, P < 0.05), an effect abolished by PEDF antibody. We conclude that PEDF mediates reduced vasculogenesis and alveolarization in neonatal hyperoxia. Bronchopulmonary dysplasia likely results from an altered balance between pro- and antiangiogenic factors.

Regulatory Interactions between Androgens, Hoxb5, and TGFβ Signaling in Murine Lung Development

Androgens enhance airway branching but delay alveolar maturation contributing to increased respiratory morbidity in prematurely born male infants. Hoxb5 protein positively regulates airway branching in developing lung. In other organs, androgen regulation intersects with Hox proteins and TGFβ-SMAD signaling, but these interactions have not been studied in the lung. We hypothesized that androgen alteration of airway branching early in lung development requires Hoxb5 expression and that these androgen-Hoxb5 interactions occur partially through regional changes in TGFβ signaling. To evaluate acute effects of androgen and TGFβ on Hoxb5, E11 whole fetal mouse lungs were cultured with dihydrotestosterone (DHT) with/without Hoxb5 siRNA or TGFβ inhibitory antibody. Chronic in utero DHT exposure was accomplished by exposing pregnant mice to DHT (subcutaneous pellet) from E11 to E18. DHTs ability to enhance airway branching and alter phosphorylated SMAD2 cellular localization was partially dependent on Hoxb5. Hoxb5 inhibition also changed the cellular distribution of SMAD7 protein. Chronic in utero DHT increased Hoxb5 and altered SMAD7 mesenchymal localization. TGFβ inhibition enhanced airway branching, and Hoxb5 protein cellular localization was more diffuse. We conclude that DHT controls lung airway development partially through modulation of Hoxb5 protein expression and that this level of regulation involves interactions with TGFβ signaling.