Fetal Lung Epithelial Cells Research Articles

Gestational Diabetes Mellitus Impedes Fetal Lung Development Through Exosome-Dependent Crosstalk Between Trophoblasts and Lung Epithelial Cells.

Fetal lung underdevelopment (FLUD) is associated with neonatal and childhood severe respiratory diseases, among which gestational diabetes mellitus (GDM) play crucial roles as revealed by recent prevalence studies, yet mechanism underlying GDM-induced FLUD, especially the role of trophoblasts, is not all known. From the perspective of trophoblast-derived exosomes, we established in vitro, ex vivo, in vivo and GDM trophoblast models. Utilizing placenta-derived exosomes (NUB-exos and GDMUB-exos) isolated from normal and GDM umbilical cord blood plasma and trophoblast-derived exosomes (NC-exos and HG-exos) isolated from HTR8/SVneo trophoblasts medium with/without high glucose treatment, we examined their effects on fetal lung development and biological functions. We found that, compared with the NUB-exos group, the exosome concentration increased in GDMUB-exos group, and the content of exosomes also changed evidenced by 61 dysregulated miRNAs. After applying these exosomes to A549 alveolar type II epithelial cells, the proliferation and biological functions were suppressed while the proportion of apoptotic cells was increased as compared to the control. In ex vivo studies, we found that GDMUB-exos showed significant suppression on the growth of the fetal lung explants, where the number of terminal buds and the area of explant surface decreased and shrank. Besides, the expression of Fgf10, Vegfa, Flt-1, Kdr and surfactant proteins A, B, C, and D was downregulated in GDMUB-exos group, whilst Sox9 was upregulated. For in vivo studies, we found significant suppression of fetal lung development in GDMUB-exos group. Importantly, we found consistent alterations when we used NC-exos and HG-exos, suggesting a dominant role of trophoblasts in placenta-derived exosome-induced FLUD. In conclusion, GDM can adversely affect trophoblasts and alter exosome contents, causing crosstalk disorder between trophoblasts and fetal lung epithelial cells and finally leading to FLUD. Findings of this study will shine insight into the theoretical explanation for the pathogenesis of FLUD.

ODP558 Steroid Receptor Coactivators Promote Non-Small Cell Lung Cancer Tumorigenesis by Inhibiting Differentiation Factors, C/EBPα and NF-κB

Abstract The steroid receptor coactivator (SRC) family comprises three members, SRC-1 (NCOA1), SRC-2 (NCOA2), and SRC-3 (NCOA3). These SRC family members modulate diverse gene expression programs both by nuclear receptors and other transcription factors to drive physiological and pathophysiological processes. Notably, SRC-1, -2 and -3 also were found to be involved in different aspects of tumorigenesis. SRC-1 and SRC-3 overexpression has been observed in endometrial, ovarian, prostate, colorectal, gastric, lung, pancreatic and liver cancers. SRC-2 expression is elevated in patients with metastatic prostate cancer. This suggests that SRC family members are involved in signaling pathways critical for cancer cell proliferation and tumorigenesis. In the present study, we have begun to elucidate the mechanisms by which SRC members play a role in non-small cell lung cancer (NSCLC). We used the potent small-molecule SRC inhibitor, SI-2, which significantly impedes primary tumor growth in a breast cancer mouse model [1]. SI-2 selectively reduces cellular protein levels of SRC-1, SRC-2 and SRC-3 post transcriptionally. We observed that treatment of the NSCLC lines A549 and H1299 with SI-2 significantly induced protein expression of CCAAT/enhancer-binding protein-α (C/EBPα). C/EBPα is a basic leucine zipper transcription factor that is highly expressed in differentiated fetal lung epithelial cells, where it controls differentiation-dependent gene expression and inhibits cell proliferation. C/EBPα was found to be inactivated in various types of cancers, providing evidence for a possible tumor suppressor function of the C/EBPα (CEBPA) gene. We found that activation of C/EBPα in SI-2 treated NSCLC (A549 and H1299) was associated with a decline in the levels of histone deacetylases, HDAC-2 and-4. This suggests that inhibiting SRC family members in lung cancer cells causes reactivation of the epigenetically suppressed CEBPA gene by inhibiting histone deacetylase expression. We also observed that SI-2 increased expression of nuclear factor-κB (NF-κB) p65 protein in both A549 and H1299 cells. Previously, we found that NF-κB activates signaling pathways leading to enhanced fetal lung epithelial cell differentiation by decreasing HDAC-2 and -4 expression and binding to promoters of differentiation-inducing genes. Overall, we speculate that the SRC inhibitor, SI-2, blocks NSCLC proliferation and tumorigenesis by enhancing histone acetylation and transcriptional activation of putative tumor suppressors, C/EBPα and NF-κB, to increase lung epithelial cell differentiation.

Mechanical properties of the premature lung: From tissue deformation under load to mechanosensitivity of alveolar cells.

Many preterm infants require mechanical ventilation as life-saving therapy. However, ventilation-induced overpressure can result in lung diseases. Considering the lung as a viscoelastic material, positive pressure inside the lung results in increased hydrostatic pressure and tissue compression. To elucidate the effect of positive pressure on lung tissue mechanics and cell behavior, we mimic the effect of overpressure by employing an uniaxial load onto fetal and adult rat lungs with different deformation rates. Additionally, tissue expansion during tidal breathing due to a negative intrathoracic pressure was addressed by uniaxial tension. We found a hyperelastic deformation behavior of fetal tissues under compression and tension with a remarkable strain stiffening. In contrast, adult lungs exhibited a similar response only during compression. Young’s moduli were always larger during tension compared to compression, while only during compression a strong deformation-rate dependency was found. In fact, fetal lung tissue under compression showed clear viscoelastic features even for small strains. Thus, we propose that the fetal lung is much more vulnerable during inflation by mechanical ventilation compared to normal inspiration. Electrophysiological experiments with different hydrostatic pressure gradients acting on primary fetal distal lung epithelial cells revealed that the activity of the epithelial sodium channel (ENaC) and the sodium-potassium pump (Na,K-ATPase) dropped during pressures of 30 cmH2O. Thus, pressures used during mechanical ventilation might impair alveolar fluid clearance important for normal lung function.

Albumin Stimulates Epithelial Na+ Transport and Barrier Integrity by Activating the PI3K/AKT/SGK1 Pathway.

Albumin is a major serum protein and is frequently used as a cell culture supplement. It is crucially involved in the regulation of osmotic pressure and distribution of fluid between different compartments. Alveolar epithelial Na+ transport drives alveolar fluid clearance (AFC), enabling air breathing. Whether or not albumin affects AFC and Na+ transport is yet unknown. We therefore determined the acute and chronic effects of albumin on Na+ transport in fetal distal lung epithelial (FDLE) cells and the involved kinase pathways. Chronic BSA treatment strongly increased epithelial Na+ transport and barrier integrity in Ussing chambers. BSA did not elevate mRNA expression of Na+ transporters in FDLE cells after 24 h. Moreover, acute BSA treatment for 45 min mimicked the chronic effects. The elevated Na+ transport was caused by an increased maximal ENaC activity, while Na,K-ATPase activity remained unchanged. Acute and chronic BSA treatment lowered membrane permeability, confirming the increased barrier integrity observed in Ussing chambers. Western blots demonstrated an increased phosphorylation of AKT and SGK1, and PI3K inhibition abolished the stimulating effect of BSA. BSA therefore enhanced epithelial Na+ transport and barrier integrity by activating the PI3K/AKT/SGK1 pathway.

Autophagy Is Impaired in Fetal Hypoplastic Lungs and Rescued by Administration of Amniotic Fluid Stem Cell Extracellular Vesicles.

Rationale: Pulmonary hypoplasia secondary to congenital diaphragmatic hernia is characterized by reduced branching morphogenesis, which is responsible for poor clinical outcomes. Administration of amniotic fluid stem cell extracellular vesicles (AFSC-EVs) rescues branching morphogenesis in rodent fetal models of pulmonary hypoplasia. Herein, we hypothesized that AFSC-EVs exert their regenerative potential by affecting autophagy, a process required for normal lung development. Objectives: To evaluate autophagy in hypoplastic lungs throughout gestation and establish whether AFSC-EV administration improves branching morphogenesis through autophagy-mediated mechanisms. Methods: EVs were isolated from c-kit+ AFSC-conditioned medium by ultracentrifugation and characterized for size, morphology, and EV markers. Branching morphogenesis was inhibited in rat fetuses by nitrofen administration to dams and in human fetal lung explants by blocking RAC1 activity with NSC23766. The expression of autophagy activators (BECN1 and ATG5) and adaptor (SQSTM1/p62) was analyzed invitro (rat and human fetal lung explants) and invivo (rat fetal lungs). Mechanistic studies on rat fetal primary lung epithelial cells were conducted using inhibitors for microRNA-17 and -20a contained in the AFSC-EV cargo and known to regulate autophagy. Measurements and Main Results: Rat and human models of fetal pulmonary hypoplasia showed reduced autophagy mainly at pseudoglandular and canalicular stages. AFSC-EV administration restored autophagy in both pulmonary hypoplasia models by transferring miR-17∼92 cluster members contained in the EV cargo. Conclusions: AFSC-EV treatment rescues branching morphogenesis partly by restoring autophagy through microRNA cargo transfer. This study enhances our understanding of pulmonary hypoplasia pathogenesis and creates new opportunities for fetal therapeutic intervention in congenital diaphragmatic hernia babies.

Epidermal growth factor strongly affects epithelial Na+ transport and barrier function in fetal alveolar cells, with minor sex-specific effects

Male sex remains an independent risk factor for respiratory distress syndrome (RDS) in preterm infants. Insufficient Na+ transport-mediated alveolar fluid clearance contributes to RDS development and we previously demonstrated sex-specific differences in Na+ transport. The epidermal growth factor (EGF) is important during fetal lung development with possible influence on Na+ transport. Sex-specific effects of EGF during surfactant synthesis were shown. We thus determined whether EGF exerts sex-specific effects on Na+ transport in fetal alveolar cells. We analyzed sex-specific fetal distal lung epithelial (FDLE) cells exposed to EGF and related ligands with Ussing chambers, RT-qPCR and Western blots. EGF strongly reduced the epithelial Na+ channel (ENaC) mRNA levels in both male and female FDLE cells. This was corroborated by a markedly reduced ENaC activity, while amiloride-insensitive pathways as well as barrier function were raised by EGF. In contrast to chronic effects, acute effects of EGF were sex-specific, because Na+ transport was reduced only in males. AKT phosphorylation was elevated only in female cells, while pERK1/2 was increased in both male and female cells. EGF showed certain sex- and time-dependent effects in FDLE cells. Nevertheless, the results suggest that EGF is an unlikely cause for the sex-specific differences in Na+ transport.

Role of NRF2 in immune modulator expression in developing lung.

After birth, the alveolar epithelium is exposed to environmental pathogens and high O2 tensions. The alveolar type II cells may protect this epithelium through surfactant production. Surfactant protein, SP-A, an immune modulator, is developmentally upregulated in fetal lung with surfactant phospholipid synthesis. Herein, we observed that the redox-regulated transcription factor, NRF2, and co-regulated C/EBPβ and PPARγ, were markedly induced during cAMP-mediated differentiation of cultured human fetal lung (HFL) epithelial cells. This occurred with enhanced expression of immune modulators, SP-A, TDO2, AhR, and NQO1. Like SP-A, cAMP induction of NRF2 was prevented when cells were exposed to hypoxia. NRF2 knockdown inhibited induction of C/EBPβ, PPARγ, and immune modulators. Binding of endogenous NRF2 to promoters of SP-A and other immune modulator genes increased during HFL cell differentiation. In mouse fetal lung (MFL), a developmental increase in Nrf2, SP-A, Tdo2, Ahr, and Nqo1 and decrease in Keap1 occurred from 14.5 to 18.5dpc. Developmental induction of Nrf2 in MFL was associated with increased nuclear localization of NF-κB p65, a decline in p38 MAPK phosphorylation, increase in the MAPK phosphatase, DUSP1, induction of the histone acetylase, CBP, and decline in the histone deacetylase, HDAC4. Thus, together with surfactant production, type II cells protect the alveolar epithelium through increased expression of NRF2 and immune modulators to prevent inflammation and oxidative stress. Our findings further suggest that lung cancer cells have usurped this developmental pathway to promote immune tolerance and enhance survival.

Abstract PO033: Regulation of immune checkpoint proteins in NSCLC adenocarcinoma

Abstract Lung cancer is the leading cause of cancer-related deaths in both men and women. Approximately 85% of lung cancers are non-small cell lung cancer (NSCLC), with adenocarcinoma (adenoCa) being the main subtype. Well-defined mutational signatures of various lung cancer subtypes have been exploited for targeted therapies, but cancer cells often acquire resistance to these treatments, which could be mediated by the inflammatory tumor microenvironment (TME). Inflammation within the TME is known to promote cancer progression and therapy resistance. The inflammatory cytokine, interleukin-1 beta (IL-1β) is elevated in the TME of several cancers and correlates with poor outcomes. In NSCLC patients, serum IL-1β is elevated relative to normal subjects; within the same patient, IL-1β is elevated in the tumor relative to adjacent non-tumor tissue. Significantly, the Canakinumab Anti-inflammatory Thrombosis Outcomes Study (CANTOS) showed that blockade of IL-1 signaling could drastically reduce lung cancer incidence and mortality. Interestingly, we observed that NSCLC adenoCa cell lines H1838, H2228, H2347, HCC4150 and HCC827 have significantly higher basal expression of the Type 1 IL-1 receptor (IL-1R1), relative to immortalized normal Human Bronchial Epithelial Cell (HBEC-3KT) and Human Small Airway Epithelial Cell (HSAEC-1KT) lines, suggesting that adenoCa cells are primed to respond to IL-1 signaling. Cancer cells can promote immunosuppression through the expression of immune checkpoint proteins (ICPs). Previously, we identified several ICPs, including programmed death-ligand 1 (PD-L1), tryptophan 2,3-dioxygenase 2 (TDO2), indoleamine 2, 3-dioxygenase 1 (IDO1) and growth differentiation factor 15 (GDF15), that were developmentally upregulated by transcription factor nuclear factor erythroid 2-related factor 2 (NRF2/NFE2L2) in human fetal lung epithelial cells during differentiation in culture. Importantly, genomic studies have revealed that >20% of lung adenoCa harbor mutations in NRF2 or its inhibitor KEAP1, which cause NRF2 activation. Lung adenoCa with signatures of increased NRF2 activation manifest reduced immune cell infiltration, suggesting that increased expression of NRF2 might create a more permissive immune cell environment, resulting in evasion and tolerance to the host immune system. Given that IL-1β induces ROS which can activate NRF2, we hypothesized that IL-1β present in the TME may enhance expression of NRF2 and ICPs in NSCLC adenoCa. We found that increased expression of ICPs in adenoCa cell lines H1838, H2228, H2347, HCC4150 and HCC827 compared to HBEC-3KT and HSAEC-1KT cells was correlated with significantly increased expression of NRF2. When these adenoCa cell lines were treated with IL-1β, we observed a marked induction in NRF2 and ICP mRNA and protein levels. Taken together our studies indicate that NSCLC adenoCa cells are primed to respond to IL-1 signaling which induces NRF2 and ICPs to promote tumor cell survival through immunosuppression. Citation Format: Afshan Fathima Nawas, Ritu Mishra, Carole R. Mendelson. Regulation of immune checkpoint proteins in NSCLC adenocarcinoma [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO033.

Paracrine stimulation of perinatal lung functional and structural maturation by mesenchymal stem cells

BackgroundMesenchymal stem cells (MSCs) were shown to harbor therapeutic potential in models of respiratory diseases, such as bronchopulmonary dysplasia (BPD), the most common sequel of preterm birth. In these studies, cells or animals were challenged with hyperoxia or other injury-inducing agents. However, little is known about the effect of MSCs on immature fetal lungs and whether MSCs are able to improve lung maturity, which may alleviate lung developmental arrest in BPD.MethodsWe aimed to determine if the conditioned medium (CM) of MSCs stimulates functional and structural lung maturation. As a measure of functional maturation, Na+ transport in primary fetal distal lung epithelial cells (FDLE) was studied in Ussing chambers. Na+ transporter and surfactant protein mRNA expression was determined by qRT-PCR. Structural maturation was assessed by microscopy in fetal rat lung explants.ResultsMSC-CM strongly increased the activity of the epithelial Na+ channel (ENaC) and the Na,K-ATPase as well as their mRNA expression. Branching and growth of fetal lung explants and surfactant protein mRNA expression were enhanced by MSC-CM. Epithelial integrity and metabolic activity of FDLE cells were not influenced by MSC-CM. Since MSC’s actions are mainly attributed to paracrine signaling, prominent lung growth factors were blocked. None of the tested growth factors (VEGF, BMP, PDGF, EGF, TGF-β, FGF, HGF) contributed to the MSC-induced increase of Na+ transport. In contrast, inhibition of PI3-K/AKT and Rac1 signaling reduced MSC-CM efficacy, suggesting an involvement of these pathways in the MSC-CM-induced Na+ transport.ConclusionThe results demonstrate that MSC-CM strongly stimulated functional and structural maturation of the fetal lungs. These effects were at least partially mediated by the PI3-K/AKT and Rac1 signaling pathway. Thus, MSCs not only repair a deleterious tissue environment, but also target lung cellular immaturity itself.

Glucocorticoids Equally Stimulate Epithelial Na+ Transport in Male and Female Fetal Alveolar Cells.

Preterm infants frequently suffer from respiratory distress syndrome (RDS), possibly due to lower expression of epithelial Na+ channels (ENaC). RDS incidence is sex-specific, affecting males almost twice as often. Despite the use of antenatal glucocorticoids (GCs), the sex difference persists. It is still controversial whether both sexes benefit equally from GCs. We previously showed that Na+ transport is higher in female compared with male fetal distal lung epithelial (FDLE) cells. Since GCs increase Na+ transport, we hypothesized that their stimulating effect might be sex-specific. We analyzed FDLE cells with Ussing chambers and RT-qPCR in the presence or absence of fetal serum. In serum-free medium, GCs increased the ENaC activity and mRNA expression, independent of sex. In contrast, GCs did not increase the Na+ transport in serum-supplemented media and abolished the otherwise observed sex difference. Inhibition of the GC receptor in the presence of serum did not equalize Na+ transport between male and female cells. The GC-induced surfactant protein mRNA expression was concentration and sex-specific. In conclusion, female and male FDLE cells exhibit no sex difference in response to GCs with regard to Na+ transport, and GR activity does not contribute to the higher Na+ transport in females.

Effect of Cell Age on Uptake and Toxicity of Nanoparticles: The Overlooked Factor at the Nanobio Interface.

Clinical translation of nanotechnologies has limited success, at least in part, due to the existence of several overlooked factors on the nature of the nanosystem (e.g., physicochemical properties of nanoparticles), nanobio interfaces (e.g., protein corona composition), and the cellular characteristics (e.g., cell type). In the past decade, several ignored factors including personalized and disease-specific protein corona (a layer of formed biomolecules at the surface of nanoparticles upon their entrance into a biological fluid), incubating temperature, local temperature gradient, cell shape, and cell sex has been introduced. Here, it was hypothesized and validated cell age as another overlooked factor in the field of nanomedicine. To test our hypothesis, cellular toxicity and uptake profiles of our model nanoparticles (i.e., PEGylated quantum dots, QDs) were probed in young and senescent cells (i.e., IMR90 fibroblast cells from human fetal lung and CCD841CoN epithelial cells from human fetal colon) and the outcomes revealed substantial dependency of cell-nanoparticles interactions to the cell age. For example, it was observed that the PEGylated QDs were acutely toxic to senescent IMR90 and CCD841CoN cells, leading to lysosomal membrane permeabilization which caused cell necrosis; in contrast, the young cells were resilient to the exact same amount of QDs and the same incubation time. It was also found that the formation of protein corona could delay the QDs' toxicity on senescent cells. These findings suggest that the cellular aging process have a capacity to cause deteriorative effects on their organelles and normal functions. The outcomes of this study suggest the proof-of-concept that cell age may have critical role in biosystem responses to nanoparticle technologies. Therefore, the effect of cell age should be carefully considered on the nanobio interactions and the information about cellular age (e.g., passage number and age of the cell donor) should be included in the nanomedicine papers to facilitate clinical translation of nanotechnologies and to help scientists to better design and produce safe and efficient diagnostic/therapeutic age-specific nanoparticles.

MON-315 Transcription Factor NRF2 May Serve as a Master Regulator of Immune Modulation and Lung Cancer Progression

Lung cancer contributes to the highest rates of cancer-related deaths world-wide. Approximately 80% of lung cancers are non-small cell lung cancers (NSCLC), and adenocarcino­mas (adenoCa) comprise about half of these. Importantly, >20% of lung adenoCa harbor mutations in transcription factor NRF2 and its inhibitor KEAP1 that promote NRF2 activation. Lung cancers with increased NRF2 levels are highly aggressive and have a poor prognosis. However, the mechanisms where­by NRF2 promotes lung cancer growth and progression are poorly understood. NRF2, a ‘primary mediator of cellular adaptation to oxida­tive stress,’ binds to promoters of anti-oxidant genes (e.g. HMOX1, NQO1), which have anti-inflammatory and immune modulatory activity. Hypothesis: NRF2 serves a crucial role in lung cancer progression by enhancing pro­duction of key immune mod­ulators that promotes immunosuppression and tumor progression. Objectives: To assess the roles of NRF2 on cAMP regulation of immune modulator expression in primary cultures of human fetal lung (HFL) epithelial cells and in human adenoCa cell lines Results: Using primary cultures of HFL epithelial cells and the human adenoCa cell lines, HCC4150 and H2347 (obtained from Dr. John Minna), we made the intri­guing discovery that cAMP markedly and coordinately induced expression of NRF2, its co-regu­lated transcriptional targets, C/EBPβ and PPARγ, the immune mod­u­la­tors, surfactant protein-A, HMOX1, GDF15, PD-L1 and TDO2, which catalyzes metab­olism of trypto­phan to kynurenine, and the kynurenine receptor, AhR. Furthermore, siRNA-mediated NRF2 knockdown inhibited C/EBPβ and PPARγ induction and blocked cAMP induction of immune modulators in both the HFL epithelial cells and adenoCa cell lines. ChIP-qPCR revealed that cAMP-induced binding of endogenous NRF2 to the promoters of all of these genes. Importantly, lung adenoCa with signatures of increased NRF2 activation manifest reduced immune cell infiltration, suggesting that NRF2 may create a more permissive immune environment. Accordingly, all of these immune modulators are known to promote survival of tumor metas­tases. Previous studies on triple-negative breast cancer cells indicated that the TDO2-AhR signaling axis mediated anoikis resistance, proliferation, migration, and invasion in culture, as well as increased metastatic capacity of xenografts in mice. Conclusions: Collectively our findings suggest that NRF2, acting with C/EBPβ and PPARγ, may serve a crucial role in lung cancer progression by enhancing pro­duction of key immune mod­ulators that promote tumor progression. We anticipate that our studies will reveal other NRF2-regulated pathways in NSCLC pathogenesis that will lead to develop­ment of novel and effective strategies for treating this devastating disease.

Acid Sphingomyelinase Inhibition Attenuates Cell Death in Mechanically Ventilated Newborn Rat Lung.

Premature infants subjected to mechanical ventilation (MV) are prone to lung injury that may result in bronchopulmonary dysplasia. MV causes epithelial cell death and halts alveolar development. The exact mechanism of MV-induced epithelial cell death is unknown. To determine the contribution of autophagy to MV-induced epithelial cell death in newborn rat lungs. Newborn rat lungs and fetal rat lung epithelial (FRLE) cells were exposed to MV and cyclic stretch, respectively, and were then analyzed by immunoblotting and mass spectrometry for autophagy, apoptosis, and bioactive sphingolipids. Both MV and stretch first induce autophagy (ATG 5-12 [autophagy related 5-12] and LC3B-II [microtubule-associated proteins 1A/1B light chain 3B-II] formation) followed by extrinsic apoptosis (cleaved CASP8/3 [caspase-8/3] and PARP [poly(ADP-ribose) polymerase] formation). Stretch-induced apoptosis was attenuated by inhibiting autophagy. Coimmunoprecipitation revealed that stretch promoted an interaction between LC3B and the FAS (first apoptosis signal) cell death receptor in FRLE cells. Ceramide levels, in particular C16 ceramide, were rapidly elevated in response to ventilation and stretch, and C16 ceramide treatment of FRLE cells induced autophagy and apoptosis in a temporal pattern similar to that seen with MV and stretch. SMPD1 (sphingomyelin phosphodiesterase 1) was activated by ventilation and stretch, and its inhibition prevented ceramide production, LC3B-II formation, LC3B/first apoptosis signal interaction, caspase-3 activation, and, ultimately, FLRE cell death. SMPD1 inhibition also attenuated ventilation-induced autophagy and apoptosis in newborn rats. Ventilation-induced ceramides promote autophagy-mediated cell death, and identifies SMPD1 as a potential therapeutic target for the treatment of ventilation-induced lung injury in newborns.

Developmental Decline in the MicroRNA 199a (miR-199a)/miR-214 Cluster in Human Fetal Lung Promotes Type II Cell Differentiation by Upregulating Key Transcription Factors.

The major surfactant protein, SP-A (a product of the SFTPA gene), serves as a marker of type II pneumocyte differentiation and surfactant synthesis. SFTPA expression in cultured human fetal lung (HFL) epithelial cells is upregulated by hormones that increase cyclic AMP (cAMP) and activate TTF-1/NKX2.1 and NF-κB. To further define mechanisms for type II cell differentiation and induction of SP-A, we investigated roles of microRNAs (miRNAs). Using microarray to identify differentially expressed miRNAs in HFL epithelial cells during type II cell differentiation in culture, we observed that members of the miRNA 199a (miR-199a)/miR-214 cluster were significantly downregulated during differentiation. Validated and predicted targets of miR-199a-3p/miR-199a-5p and miR-214, which serve roles in type II cell differentiation (COX-2, NF-κB p50/p65, and CREB1), and the CREB1 target, C/EBPβ, were coordinately upregulated. Accordingly, overexpression of miR-199a-5p, miR-199a-3p, or miR-214 mimics in cultured HFL epithelial cells decreased COX-2, NF-κB p50/p65, CREB1, and C/EBPβ proteins, with an associated inhibition of SP-A expression. Interestingly, overexpression of the EMT factor, ZEB1, which declines during cAMP-induced type II cell differentiation, increased pri-miR-199a and reduced the expression of the targets NF-κB/p50 and COX-2. Collectively, these findings suggest that the developmental decline in miR-199a/miR-214 in HFL causes increased expression of critical targets that enhance type II cell differentiation and SP-A expression.

Regulation of vascular signalling by nuclear Sprouty2 in fetal lung epithelial cells: Implications for co-ordinated airway and vascular branching in lung development.

Sprouty2 (Spry2) acts as a central regulator of tubular growth and branch patterning in the developing mammalian lung by controlling both magnitude and duration of growth factor signalling. To determine if this protein coordinates airway and vascular growth factor signalling, we tested the hypothesis that Spry2 links the primary cue for airway outgrowth, fibroblast growth factor-10 (FGF-10), to genomic events underpinning the expression and release of vascular endothelial growth factor-A (VEGF-A). Using primary fetal distal lung epithelial cells (FDLE) from rat, and immortalised human bronchial epithelial cells (16HBE14o-), we identified a nuclear sub-population of Spry2 which interacted with regions of the rat and human VEGF-A promoter spanning the hypoxia response element (HRE) and adjacent 3′ sites. In FDLE cultured at the PO2 of the fetal lung, FGF-10 relieved the Spry2 interaction at the HRE region by promoting clearance of a 39 kDa form and this was accompanied by histone-3 S10K14 phosphoacetylation, promoter de-methylation, hypoxia inducible factor-1α activation and VEGF-A expression. This repressive characteristic of nuclear Spry2 was relieved in 16HBE14o- by shRNA knockdown, and stable expression of mutants (C218A; C221A) that do not interact with the VEGF-A promoter HRE region. We conclude that nuclear Spry2 acts as a molecular link which co-ordinates airway and vascular growth of the cardiopulmonary system. This identifies Spry2 as a contributing determinant of design optimality in the mammalian lung.

InVitro Induction and InVivo Engraftment of Lung Bud Tip Progenitor Cells Derived from Human Pluripotent Stem Cells.

SummaryThe current study aimed to understand the developmental mechanisms regulating bud tip progenitor cells in the human fetal lung, which are present during branching morphogenesis, and to use this information to induce a bud tip progenitor-like population from human pluripotent stem cells (hPSCs) in vitro. We identified cues that maintained isolated human fetal lung epithelial bud tip progenitor cells in vitro and induced three-dimensional hPSC-derived organoids with bud tip-like domains. Bud tip-like domains could be isolated, expanded, and maintained as a nearly homogeneous population. Molecular and cellular comparisons revealed that hPSC-derived bud tip-like cells are highly similar to native lung bud tip progenitors. hPSC-derived epithelial bud tip-like structures survived in vitro for over 16 weeks, could be easily frozen and thawed, maintained multilineage potential, and successfully engrafted into the airways of immunocompromised mouse lungs, where they persisted for up to 6 weeks and gave rise to several lung epithelial lineages.

Cytotoxic activity of T-2 toxin for immortalized cultures of fetal calf lung epithelial cells

Cytotoxic activity of T-2 toxin for immortalized cultures of fetal calf lung epithelial cells

Sex-specific effects of sex steroids on alveolar epithelial Na+ transport.

Alveolar fluid clearance mediates perinatal lung transition to air breathing in newborn infants, which is accomplished by epithelial Na+ channels (ENaC) and Na-K-ATPase. Male sex represents a major risk factor for developing respiratory distress, especially in preterm infants. We previously showed that male sex is associated with reduced epithelial Na+ transport, possibly contributing to the sexual dimorphism in newborn respiratory distress. This study aimed to determine sex-specific effects of sex steroids on epithelial Na+ transport. The effects of testosterone, 5α-dihydrotestosterone (DHT), estradiol, and progesterone on Na+ transport and Na+ channel expression were determined in fetal distal lung epithelial (FDLE) cells of male and female rat fetuses by Ussing chamber and mRNA expression analyses. DHT showed a minor effect only in male FDLE cells by decreasing epithelial Na+ transport. However, flutamide, an androgen receptor antagonist, did not abolish the gender imbalance, and testosterone lacked any effect on Na+ transport in male and female FDLE cells. In contrast, estradiol and progesterone increased Na+ transport and Na+ channel expression especially in females, and prevented the inhibiting effect of DHT in males. Estrogen receptor inhibition decreased Na+ channel expression and eliminated the sex differences. In conclusion, female sex steroids stimulate Na+ transport especially in females and prevent the inhibitory effect of DHT in males. The ineffectiveness of testosterone suggests that Na+ transport is largely unaffected by androgens. Thus, the higher responsiveness of female cells to female sex steroids explains the higher Na+ transport activity, possibly leading to a functional advantage in females.

CRISPLD2 (LGL1) inhibits proinflammatory mediators in human fetal, adult, and COPD lung fibroblasts and epithelial cells.

Chronic lung disease of prematurity/bronchopulmonary dysplasia (BPD) is the leading cause of perinatal morbidity in developed countries. Inflammation is a prominent finding. Currently available interventions have associated toxicities and limited efficacy. While BPD often resolves in childhood, survivors of preterm birth are at risk for acquired respiratory disease in early life and are more likely to develop chronic obstructive pulmonary disease (COPD) in adulthood. We previously cloned Crispld2 (Lgl1), a glucocorticoid‐regulated mesenchymal secretory protein that modulates lung branching and alveogenesis through mesenchymal–epithelial interactions. Absence of Crispld2 is embryonic lethal. Heterozygous Crispld2+/− mice display features of BPD, including distal airspace enlargement, disruption of elastin, and neonatal lung inflammation. CRISPLD2 also plays a role in human fetal lung fibroblast cell expansion, migration, and mesenchymal–epithelial signaling. This study assessed the effects of endogenous and exogenous CRISPLD2 on expression of proinflammatory mediators in human fetal and adult (normal and COPD) lung fibroblasts and epithelial cells. CRISPLD2 expression was upregulated in a lipopolysaccharide (LPS)‐induced human fetal lung fibroblast line (MRC5). LPS‐induced upregulation of the proinflammatory cytokines IL‐8 and CCL2 was exacerbated in MRC5‐CRISPLD2 knockdown cells. siRNA suppression of endogenous CRISPLD2 in adult lung fibroblasts (HLFs) led to augmented expression of IL‐8, IL‐6, CCL2. LPS‐stimulated expression of proinflammatory mediators by human lung epithelial HAEo‐ cells was attenuated by purified secretory CRISPLD2. RNA sequencing results from HLF‐CRISPLD2 knockdown suggest roles for CRISPLD2 in extracellular matrix and in inflammation. Our data suggest that suppression of CRISPLD2 increases the risk of lung inflammation in early life and adulthood.

Role of epithelial sodium channels in the regulation of lung fluid homeostasis.

In utero, fetal lung epithelial cells actively secrete Cl(-) ions into the lung air spaces while Na(+) ions follow passively to maintain electroneutrality. This process, driven by an electrochemical gradient generated by the Na(+)-K(+)-ATPase, is responsible for the secretion of fetal fluid that is essential for normal lung development. Shortly before birth, a significant upregulation of amiloride-sensitive epithelial channels (ENaCs) on the apical side of the lung epithelial cells results in upregulation of active Na(+) transport. This process is critical for the reabsorption of fetal lung fluid and the establishment of optimum gas exchange. In the adult lung, active Na(+) reabsorption across distal lung epithelial cells limits the degree of alveolar edema in patients with acute lung injury and cardiogenic edema. Cl(-) ions are transported either paracellularly or transcellularly to preserve electroneutrality. An increase in Cl(-) secretion across the distal lung epithelium has been reported following an acute increase in left atrial pressure and may result in pulmonary edema. In contrast, airway epithelial cells secrete Cl(-) through apical cystic fibrosis transmembrane conductance regulator and Ca(2+)-activated Cl(-) channels and absorb Na(+). Thus the coordinated action of Cl(-) secretion and Na(+) absorption is essential for maintenance of the volume of epithelial lining fluid that, in turn, maximizes mucociliary clearance and facilitates clearance of bacteria and debris from the lungs. Any factor that interferes with Na(+) or Cl(-) transport or dramatically upregulates ENaC activity in airway epithelial cells has been associated with lung diseases such as cystic fibrosis or chronic obstructive lung disease. In this review we focus on the role of the ENaC, the mechanisms involved in ENaC regulation, and how ENaC dysregulation can lead to lung pathology.