MicroRNA-214 promotes alveolarization in neonatal rat models of bronchopulmonary dysplasia via the PlGF-dependent STAT3 pathway

Background Recently, the role of several microRNAs (miRNAs or miRs) in pulmonary diseases has been described. The molecular mechanisms by which miR-214 is possibly implicated in bronchopulmonary dysplasia (BPD) have not yet been addressed. Hence, this study aimed to investigate a putative role of miR-214 in alveolarization among preterm neonates with BPD. Methods Microarray-based gene expression profiling data from BPD was employed to identify differentially expressed genes. A BPD neonatal rat model was induced by hyperoxia. Pulmonary epithelial cells were isolated from rats and exposed to hyperoxia to establish cell injury models. Gain- and loss-of-function experiments were performed in BPD neonatal rats and hyperoxic pulmonary epithelial cells. MiR-214 and PlGF expression in BPD neonatal rats, and eNOS, Bcl-2, c-myc, Survivin, α-SMA and E-cadherin expression in hyperoxic pulmonary epithelial cells were measured using RT-qPCR and Western blot analysis. The interaction between PlGF and miR-214 was identified using dual luciferase reporter gene and RIP assays. IL-1β, TNF-a, IL-6, ICAM-1 and Flt-1 expression in the rat models was measured using ELISA. Results The lung tissues of neonatal rats with BPD showed decreased miR-214 expression with elevated PlGF expression. PlGF was found to be a target of miR-214, whereby miR-214 downregulated PlGF to inactivate the STAT3 pathway. miR-214 overexpression or PlGF silencing decreased the apoptosis of hyperoxic pulmonary epithelial cells in vitro and restored alveolarization in BPD neonatal rats. Conclusion Overall, the results demonstrated that miR-214 could facilitate alveolarization in preterm neonates with BPD by suppressing the PlGF-dependent STAT3 pathway.

Long-Term Exposure to Nanosized TiO2 Triggers Stress Responses and Cell Death Pathways in Pulmonary Epithelial Cells

There is little in vitro data available on long-term effects of TiO2 exposure. Such data are important for improving the understanding of underlying mechanisms of adverse health effects of TiO2. Here, we exposed pulmonary epithelial cells to two doses (0.96 and 1.92 µg/cm2) of TiO2 for 13 weeks and effects on cell cycle and cell death mechanisms, i.e., apoptosis and autophagy were determined after 4, 8 and 13 weeks of exposure. Changes in telomere length, cellular protein levels and lipid classes were also analyzed at 13 weeks of exposure. We observed that the TiO2 exposure increased the fraction of cells in G1-phase and reduced the fraction of cells in G2-phase, which was accompanied by an increase in the fraction of late apoptotic/necrotic cells. This corresponded with an induced expression of key apoptotic proteins i.e., BAD and BAX, and an accumulation of several lipid classes involved in cellular stress and apoptosis. These findings were further supported by quantitative proteome profiling data showing an increase in proteins involved in cell stress and genomic maintenance pathways following TiO2 exposure. Altogether, we suggest that cell stress response and cell death pathways may be important molecular events in long-term health effects of TiO2.

A Custom-Made Device for Reproducibly Depositing Pre-metered Doses of Nebulized Drugs on Pulmonary Cells in vitro

The deposition of pre-metered doses (i.e., defined before and not after exposition) at the air–liquid interface of viable pulmonary epithelial cells remains an important but challenging task for developing aerosol medicines. While some devices allow quantification of the deposited dose after or during the experiment, e.g., gravimetrically, there is still no generally accepted way to deposit small pre-metered doses of aerosolized drugs or pharmaceutical formulations, e.g., nanomedicines. Here, we describe a straightforward custom-made device, allowing connection to commercially available nebulizers with standard cell culture plates. Designed to tightly fit into the approximately 12-mm opening of either a 12-well Transwell® insert or a single 24-well plate, a defined dose of an aerosolized liquid can be directly deposited precisely and reproducibly (4.8% deviation) at the air–liquid interface (ALI) of pulmonary cell cultures. The deposited dose can be controlled by the volume of the nebulized solution, which may vary in a range from 20 to 200 μl. The entire nebulization-deposition maneuver is completed after 30 s and is spatially homogenous. After phosphate-buffered saline (PBS) deposition, the viability and barrier properties transepithelial electrical resistance (TEER) of human bronchial epithelial Calu-3 cells were not negatively affected. Straightforward in manufacture and use, the device enables reproducible deposition of metered doses of aerosolized drugs to study the interactions with pulmonary cell cultures grown at ALI conditions.