American journal of physiology. Lung cellular and molecular physiology最新文献

The role of COPD susceptibility gene Hedgehog (Hh) Interacting Protein (HHIP) in lung tissue damage and abnormal repair in COPD is incompletely understood. We hypothesized that dysregulated HHIP expression affects cigarette smoke-induced epithelial damage and repair within the epithelial-mesenchymal tropic unit. HHIP expression was assessed in lung tissue and airway epithelial cells (AECs) from COPD patients and non-COPD controls. The effect of HHIP overexpression was assessed on cigarette smoke extract (CSE)-induced changes in epithelial plasticity genes, e.g. Cadherin 1 (CDH1, encoding E-cadherin) in 16HBE cells, and on epithelial-mesenchymal interactions during alveolar repair as modeled by organoid formation using distal lung-derived mesenchymal stromal cells (LMSCs) and EpCAM⁺ epithelial cells. We observed no abnormalities in HHIP protein levels in lung tissue of COPD patients, while the expression of HHIP was significantly lower in COPD-derived AECs compared to control. HHIP overexpression in 16HBE cells attenuated the CSE-induced reduction in CDH1 expression. Furthermore, overexpression of HHIP significantly suppressed Sonic hedgehog-induced GLI1 expression in control but not COPD-derived LMSCs, and resulted in formation of more and larger organoids, which was not observed for COPD-derived LMSCs. This defect was accompanied by lower expression of the growth factor FGF10 upon HHIP overexpression in COPD compared to control-derived LMSCs. Together, our data suggest a protective role of HHIP in CSE-induced airway epithelial responses and a supportive role in alveolar epithelial regeneration, which may be impaired in COPD.

The contractile mechanisms involving Calcium (Ca2+) regulation in smooth muscle cells (SMC) of small intrapulmonary veins (SPV) are not yet fully understood despite being relevant in lung physiology. Studies on vasoconstriction induced by physiological agonists have not been sufficient to understand the role of ryanodine receptors (RyRs). These calcium channels release Ca2+ from the sarcoplasmic reticulum (SR), within the contraction process of SPV. We hypothesize that RyRs act in a stimulus-dependent manner. Here, we first analyzed existing single-cell RNA sequencing datasets of human and rat lungs to evaluate the expression of RyRs in SMC and other cell types, followed by functional studies to assess SPV contraction using an ex vivo precision-cut lung slice (PCLS) model. To understand the participation of RyRs downstream in contraction pathways, different contractile agents were combined with RyRs agonists and antagonists. We demonstrated that RyRs are expressed in lung smooth muscle cells of humans and rats, participating in SPV contraction likely through the Gq-coupled protein receptor pathway.

Premature infants are at higher risk for developing chronic lung diseases especially following supplemental oxygen (hyperoxia) in early life. We previously demonstrated that moderate hyperoxia (<60% O₂) induces cellular senescence in fetal airway smooth muscle cells (fASM) and fibroblasts. However, the mechanisms underlying O₂-induced senescence are still under investigation. In this study we investigated the role of endoplasmic reticulum (ER) stress and mitochondrial dysfunction, using fASM cells exposed to 21% (normoxia) vs. ~50% O₂ (hyperoxia). Normoxia or hyperoxia-exposed fASM were treated with the ER stress inhibitor salubrinal [12.5 μM], the antioxidant MitoQ [100 nM] or the mitochondrial fission inhibitor Mdivi-1 [10 μM]. Samples were harvested at day 2, 3 and 7 and analyzed for markers of senescence, oxidative stress, ER stress response and mitochondrial dynamics using protein analysis and fluorescence microscopy. Hyperoxia enhanced senescence, upregulating multiple markers of DNA damage in particular, cyclin dependent cell cycle regulator p21, cytosolic and mitochondrial reactive oxygen species (ROS) levels, mitochondria fragmentation and anti- apoptosis Bcl-xL, while downregulating the proliferation marker Ki-67. Hyperoxia also activated all three ER stress pathways. However, the level of p21 and/or Bcl-xL was decreased in hyperoxia-exposed cells treated with the ER stress inhibitor salubrinal or the antioxidant MitoQ, but not the fission inhibitor Mdivi-1. These findings highlight the role of mitochondrial ROS and ER stress in hyperoxia-induced senescence of fASM and suggest that via mitochondrial targeted antioxidants and/or inhibitors of ER stress pathways can blunt the detrimental effects of hyperoxia in developing lung.

Vaping products contain numerous flavor chemicals that are known immunological sensitizers associated with the elicitation of immune-mediated hypersensitivity responses. Therefore, we investigated the immune response associated with sensitizing flavor chemicals in vaping liquids and focused on dendritic cell maturation as it is a crucial early cellular event of the sensitization immunological cascade. Bone morrow-derived dendritic cells (BMDDCs) were used to assess induction of dendritic cell maturation upon treatment with unique flavor vaping liquids and aerosol condensates. Female BALB/c mice were exposed 2h/day for 4 days to nicotine-free vaping aerosols containing a mixture of citral, cinnamaldehyde, dihydrocoumarin and vanillin. In vitro experiments indicate that cinnamaldehyde and citral, but not vaping liquid solvents, dihydrocoumarin and vanillin, are capable of inducing BMDDC maturation. In vivo, we observed an increase in the expression of maturation marker MHCII on conventional dendritic cells (cDCs) in the lung tissue of mice exposed to flavored vaping aerosols compared to exposure controls. Maturation was specifically observed on CD11b+ cDCs and not CD103+ cDCs. No significant changes were observed for macrophages, neutrophils, B and T lymphocytes in the lung tissue and bronchoalveolar lavage. Also, we found that regulatory T lymphocytes had decreased expression of CD25 in mice exposed to flavored vaping aerosols compared to exposure controls. These findings indicate that flavored chemicals can lead to rapid dendritic cell maturation in vitro and in vivo, representing an early cellular event related to respiratory sensitization and pulmonary disease.

Mammalian lung development depends on growth and differentiation of both endothelial and epithelial subpopulations to allow for gas exchange. Premature infants are born with developmentally immature lungs and often require supplemental oxygen (O₂) to survive. Excess O₂ can lead to oxidative stress which damages the pulmonary vasculature and contributes to bronchopulmonary dysplasia (BPD). Selenoproteins are critical for detoxifying reactive oxygen intermediates (ROI). Selenoprotein production is dependent upon adequate selenium (Se) levels. Using a model of perinatal Se deficiency in C3H/HeN mice, we assessed the impacts of Se status and postnatal O₂ exposure on lung vascular development at P14. Further, we compared the transcription of endothelial subpopulation and endothelial to mesenchymal transition markers in control and O₂-exposed lungs using RNAseq from P3 mouse lungs. Transcriptional changes identified from RNAseq were validated using qRT-PCR. Se deficiency and O₂ exposure independently decreased the number of pulmonary arterioles at P14. In addition, Se deficiency and O₂ exposure decreased transcription of the general capillary endothelial cell markers Aplnr and Ptprb. These findings support the hypothesis that Se deficiency confers susceptibility to hyperoxic pulmonary vascular maldevelopment as is seen in BPD.

The increasing use of electronic cigarettes (e-cigs) among adolescents poses significant public health risks. This study investigates the impact of e-cigs on the airway epithelial barrier, focusing on apical junctional complexes (AJCs), including tight junctions (TJs) and adherens junctions (AJs). We hypothesized that e-cigs disrupt AJCs in a mouse model, leading to increased airway barrier permeability. C57BL/6 mice were exposed to 36 mg/mL e-cig aerosols (3 puffs/min) for 1 h daily over 4 days. Bronchoalveolar lavage (BAL) fluid analysis, lung inflammation assessment, immunohistochemistry (IHC) staining, Western blotting (WB), and permeability assays were performed to evaluate the structure and function of the airway barrier. E-cig-exposed mice showed weight loss and elevated serum cotinine levels. BAL fluid analysis revealed elevated white blood cells. Histological analysis confirmed lung inflammation, whereas IHC and WB showed significant AJC disruption. Notably, claudin-2 levels were elevated in e-cig-exposed mice compared with controls. Claudin-2, known for its role in promoting permeability in "leaky" epithelia, increased alongside decreases in other TJ components, signifying structural barrier impairment. After e-cig exposure, instilling fluorescein isothiocyanate (FITC)-dextran into the airway increased serum FITC-dextran levels, indicating enhanced barrier permeability. E-cig aerosol exposure disrupts airway epithelial barrier structure and function, primarily through the disassembly of TJs and AJs. These findings suggest potential pathways for further clinical investigation into the health risks of e-cig use.NEW & NOTEWORTHY The rising use of e-cigs among youth has become a significant public health concern. This study, using a mouse model, demonstrates that exposure to e-cig aerosol leads to airway inflammation, structural damage to the airway epithelial barrier, and increased epithelial barrier permeability.

Cigarette smoke (CS) is a leading cause of chronic obstructive pulmonary disease (COPD). Here, we investigated whether the ion channel amplifier nesolicaftor rescues CS-induced mucociliary and ion channel dysfunction. As CS increases the expression of transforming growth factor-beta1 (TGF-β1), human bronchial epithelial cells (HBECs) from healthy donors were used for TGF-β1 and COPD donors (COPD-HBEC) for CS exposure experiments. CS and TGF-β1 induce mucociliary dysfunction by increasing MUC5AC and decreasing ion channel conductance important for mucus hydration. These include cystic fibrosis transmembrane conductance regulator (CFTR) and apical large-conductance, Ca²⁺-activated K⁺ (BK) channels. Nesolicaftor rescued CFTR and BK channel dysfunction, restored ciliary beat frequency (CBF), and decreased mucus viscosity and MUC5AC expression in CS-exposed COPD-HBEC. Nesolicaftor further reversed reductions in airway surface liquid (ASL) volumes, CBF, and CFTR and BK conductance, and blocked the increase in extracellular signal-regulated kinase (ERK) signaling in TGF-β1-exposed normal HBECs. Mechanistically, nesolicaftor increased, as expected, not only binding of PCBP1 to CFTR mRNA but also surprisingly to LRRC26 mRNA, which encodes the gamma subunit required for BK function. Similar to nesolicaftor, the angiotensin receptor blocker (ARB) losartan rescued TGF-β1-mediated decreases in PCBP1 binding to LRRC26 mRNA. In addition, the ARB telmisartan restored PCBP1 binding to CFTR and LRRC26 mRNAs to rescue CFTR and BK function in CS-exposed COPD-HBEC. Thus, nesolicaftor and ARBs act on the same target and were therefore neither additive nor synergistic in their actions. These data demonstrate that nesolicaftor and ARBs may provide benefits in COPD by improving ion channel function important for mucus hydration.NEW & NOTEWORTHY Cigarette smoke (CS) increases transforming growth factor-beta1 (TGF-β1) expression that causes mucociliary dysfunction by decreasing ion channel function. In our study, a CFTR amplifier (nesolicaftor) and angiotensin II receptor blockers (losartan and telmisartan) improve CS-induced ion channel dysfunction, by increasing binding of PCBP1 to CFTR and LRRC26 mRNAs. Therefore, nesolicaftor and ARBs, acting on the same target, may provide therapeutic benefits for treating smoking-related diseases.

Preterm infants born to mothers with preeclampsia, a disease of vascular dysfunction, are at increased risk for bronchopulmonary dysplasia (BPD). Endothelial cells are critical in both maintaining proper vascular function and coordinating lung development. Understanding the mechanisms contributing to BPD in the setting of preeclampsia and how preeclampsia impacts pulmonary endothelial cells (PECs) in the newborn lung are required to decrease the burden of BPD. Vitamin D has been shown to improve lung angiogenesis and lung development in inflammatory models of BPD, but its therapeutic potential in the setting of preeclampsia is unknown. We hypothesized that intraamniotic (IA) treatment with the biologically active form of vitamin D, 1,25 dihydroxyvitamin D [1,25(OH)₂D], will preserve lung growth in an experimental model of BPD induced by antenatal exposure to soluble vascular endothelial growth factor receptor-1 [sFlt-1 (soluble fms-like tyrosine kinase 1)]. Fetal rats were exposed to saline (control), sFlt-1 alone, 1,25(OH)₂D alone, or simultaneous sFlt-1 + 1,25(OH)₂D via IA injection during the late canalicular stage of lung development and delivered 2 days later. IA treatment with 1,25(OH)₂D in sFlt-1-exposed pups improved lung alveolar and vascular growth and function at 14 days of life. PECs orchestrate alveolar development, and we demonstrate that IA sFlt-1 exposure alone decreased in vitro growth and tube formation of PECs isolated from newborn pups and that PECs from pups coexposed to IA sFlt-1 and 1,25(OH)₂D demonstrated increased growth and tube formation. We conclude that IA 1,25(OH)₂D treatment improves distal lung development during sFlt-1 exposure through preservation of angiogenesis in the developing lung.NEW & NOTEWORTHY This study highlights that experimental BPD induced by intraamniotic sFlt-1 is associated with impaired growth in postnatal pulmonary endothelial cells. We demonstrate that 1,25(OH)₂D may be a therapeutic option to improve lung development through enhancement of VEGF signaling and preservation of early pulmonary endothelial growth in the newborn rat lung.