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

Dysregulated redox signaling contributes to pulmonary hypertension (PH) and vascular depletion of the redox enzyme extracellular superoxide dismutase (EC-SOD) from smooth muscle cells [EC-SOD SMC knockout (KO)] worsens chronic hypoxic PH. Given the important role of macrophages in PH, this study aimed to determine if interstitial macrophages (IMs) and their interactions with hyaluronan (HA), a component of extracellular matrix (ECM), are modulated by vascular EC-SOD. Floxed wild-type, EC-SOD SMC KO, and SOD mimetic- or vehicle-treated mice were exposed to hypobaric hypoxia [∼10% fraction of inspired oxygen ([Formula: see text])], for 4, 14, or 21 days. Using flow cytometry, we demonstrated that the transient increase in IMs at day 4 was exacerbated in EC-SOD SMC KO mice and prevented with SOD mimetic pretreatment. Highlighting the importance of targeting vascular oxidative stress in the early response to hypoxia, pretreatment with a single dose of EC-SOD mimetic decreased right ventricular systolic pressure, right ventricular hypertrophy, and small vessel muscularization at day 21. To assess IM phenotypic reprogramming in hypoxia, RNA-seq was performed on flow-sorted IMs revealing baseline proinflammatory activation and enhanced activation of vascular and ECM remodeling pathways in response to hypoxia in EC-SOD SMC KO IMs compared with controls. To further investigate the ECM remodeling response, we quantified IMs expressing the lymphatic vessel endothelial hyaluronan receptor 1 (Lyve1), and IM-hyaluronan binding. Lyve1⁺ IMs and Lyve1⁺ HA⁺ IMs were increased in response to hypoxia in EC-SOD SMC KO mice and accumulated in the perivascular space of the lung. In conclusion, vascular EC-SOD limits IM accumulation and proinflammatory profibrotic IM signaling, including perivascular accumulation of Lyve1⁺ IMs and their binding to hyaluronan.NEW & NOTEWORTHY Expression of the redox enzyme EC-SOD limits PH severity. Using vascular-selective EC-SOD depletion and SOD mimetic treatment in chronic hypoxic PH, we demonstrated that EC-SOD limits the hypoxia-induced accumulation of IMs. IMs from mice with low vascular EC-SOD were proinflammatory at baseline and enhanced ECM remodeling pathway activation in response to hypoxia. We identified Lyve1⁺ IMs as a perivascular, ECM-interacting subset that accumulate in hypoxia and could contribute to vascular remodeling in PH.

Type 2 inflammation and epithelial-to-mesenchymal transitions (EMTs) play critical roles in airway repair after damage from allergens or parasites. The matricellular protein periostin (POSTN) has increased expression in inflammatory conditions and has been implicated in fibrosis and EMT, suggesting a role in airway repair. This study investigates the role of periostin in airway epithelial and lung fibroblast wound repair using an in vitro wound model. Our results demonstrate that the type 2 cytokine IL-13 induces periostin secretion from primary human airway epithelial basal cells. Periostin knockdown in human airway epithelial cells (HAEs) and human lung fibroblasts (HLFs) impairs wound closure, indicating that periostin is required for airway repair. In a coculture model of HAE and HLFs, fibroblast-secreted POSTN is required for airway epithelial wound repair, suggesting that periostin is involved in paracrine signaling between the two cell types. These findings highlight periostin's critical function in epithelial and fibroblast-mediated wound repair, suggesting its potential as a therapeutic target for diseases characterized by aberrant wound healing and fibrosis, such as asthma and idiopathic pulmonary fibrosis.NEW & NOTEWORTHY This article highlights the critical role of periostin (POSTN) in airway epithelial and fibroblast-mediated wound repair. Moreover, the study reveals a paracrine signaling loop between airway epithelial basal cells and lung fibroblasts, emphasizing periostin's therapeutic potential for diseases like asthma and idiopathic pulmonary fibrosis.

Tumor necrosis factor-α (TNFα) is a pro-inflammatory cytokine, which mediates acute inflammatory effects in response to allergens, pollutants, and respiratory infections. Previously, we reported that TNFα increased maximum O₂ consumption rate (OCR) and mitochondrial volume density (MVD) in human airway smooth muscle (hASM) cells. However, TNFα decreased maximum OCR when normalized to mitochondrial volume. In addition, TNFα altered mitochondrial distribution and motility within hASM cells. Although high-resolution respirometry is valuable for assessing mitochondrial function, it overlooks mitochondrial structural and functional heterogeneity within cells. Therefore, a direct measurement of cellular mitochondrial function provides valuable information. Previously, we developed a confocal-based quantitative histochemical technique to determine the maximum velocity of the succinate dehydrogenase (SDH) reaction (SDH_max) in single cells and observed that cellular SDH_max corresponds with MVD. Therefore, we hypothesized that TNFα decreases SDH_max per mitochondrion in hASM cells. The hASM cells were treated with TNFα (20 ng/mL, 6 h, and 24 h) or untreated (time-matched control). Using three-dimensional (3-D) confocal imaging of labeled mitochondria and a concentric shell method for analysis, we quantified MVD, mitochondrial complexity index (MCI) and SDH_max relative to the nuclear membrane. Within each shell, SDH_max and MVD peaked in the perinuclear compartments and decreased toward the distal compartments of the cell. When normalized to mitochondrial volume, SDH_max decreased in the perinuclear compartments compared with distal compartments. TNFα caused a significant shift in mitochondrial morphometry and function compared to control. In conclusion, mitochondria within individual cells exhibit distinct morphological and functional heterogeneity, which is disrupted during acute inflammation.NEW & NOTEWORTHY Mitochondria show context-specific heterogeneity in their morphometry. Previously, we reported that acute TNFα exposure increased O₂ consumption rate (OCR) and mitochondrial volume density, but decreased OCR per mitochondrion. TNFα also altered mitochondrial distribution and motility. To assess TNFα-mediated subcellular mitochondrial structural and functional heterogeneity, we used a confocal-based quantitative histochemical technique to determine the maximum velocity of succinate dehydrogenase reaction. Our findings highlight that mitochondria within cells exhibit functional heterogeneity, which is disrupted during inflammation.

Lumacaftor, the corrector of Orkambi, enhances the processing of F508del cystic fibrosis transmembrane conductance regulator (CFTR), but its impact on the channel activity of rescued F508del CFTR (rF508del) is unclear. Using an electrode-based, real-time iodide efflux assay performed at room temperature, acute exposure to lumacaftor was shown to increase the processing of F508del CFTR without a proportional increase in channel activity in a CFBE41o-cell line stably expressing F508del CFTR (CFBE-DF). A similar effect was not observed on wild-type CFTR in a HEK293 cell line. At 37°C, rF508del channel activity is significantly inhibited in CFBE-DF cells by acute exposure to 5 µM lumacaftor, but not to 5 µM tezacaftor or 1 µM elexacaftor, the two correctors of Trikafta. Lumacaftor's inhibitory effect was characterized by a major left shift of the peak channel activity relative to the peak CFTR processing in the dose-response chart, which is absent for tezacaftor or elexacaftor. Ussing chamber analysis on polarized CFBE-DF cells reveals an inhibitory effect for lumacaftor on the forskolin- and ivacaftor-induced change in short-circuit current. Single channel patch clamp on HEK-DF cells shows that acute application of cytosolic lumacaftor significantly decreases rF508del channel open probability. Taken together, despite its strong corrector activity, lumacaftor inhibits rF508del channel activity, compromising the degree of functional rescue. This effect may contribute to the limited clinical efficacy of Orkambi.NEW & NOTEWORTHY Small-molecule correctors bind to F508del cystic fibrosis transmembrane conductance regulator (CFTR) and restore its trafficking to the plasma membrane to function as an anion channel. Despite its high efficacy as a corrector, lumacaftor inhibits the channel opening of rescued F508del CFTR, making it a weak CFTR modulator. The current work highlights the impact of CFTR correctors on the channel activity of rescued F508del CFTR as an important variable in the efficacy of modulator therapy.

The air-liquid interface (ALI) culture is an important tool in pulmonary research as it models the physiological lung where the epithelium is apically exposed to air and basally to the endothelium and interstitium. Although there is an abundance of research that uses primary human bronchial epithelial cells (HBECs) to study larger airways, small airway epithelial cells (SAECs) are an untapped resource in comparison. Primary SAECs are a valuable cell population as they enable the study of pathologies in the bronchioles and are also a favorable surrogate for primary alveolar epithelial cells, which are invasive to collect from patients. Currently, there are limited resources on how to culture and differentiate SAECs at the ALI. Here, we provide an optimized, detailed protocol to address this knowledge gap. Key culture conditions that determine the quality and uniformity of differentiated SAECs include cell passage number, pH changes caused by media exhaustion and incubator CO₂, seeding density, and collagen coating of the expansion flask and inserts. We also describe a FITC-dextran permeability assay to measure SAEC barrier integrity both as a pretest to select uniform wells with strong barrier integrity before an experiment and as a post-test to evaluate treatment effects afterward. The utility of the differentiated SAEC ALI model to ask biologically relevant questions is demonstrated by increased cytokine (IL-8, MIF, and CXCL-10) production and/or epithelial damage following exposure to cigarette smoke, lipopolysaccharide (LPS) or poly(I:C).NEW & NOTEWORTHY SAECs are not commonly used in pulmonary research, and this is reflected in a lack of literature on both SAEC primary research and methodological reports. Primary SAECs are an important resource as they enable the study of the small airways, which are implicated in a variety of pulmonary diseases, including chronic obstructive pulmonary disease (COPD). The detailed protocol described here bridges the knowledge gap on how to successfully differentiate primary human SAECs at the ALI.

l-cysteine ethyl ester (l-CYSee) overcomes adverse effects elicited by systemic injection of morphine on ventilatory parameters and arterial blood-gas chemistry in rats. l-CYSee or l-cysteine, resulting from the deesterification of l-CYSee, may enter enzymatic cascades that produce the ventilatory stimulant molecule, hydrogen sulfide (H₂S). dl-propargylglycine (dl-PROP) is an inhibitor of cystathionine-γ-lyase (CSE)-mediated conversion of l-cysteine to H₂S and has been widely used in vivo. We examined whether l-CYSee (2 injections × 500 µmol/kg, IV)-induced reversal of the changes in ventilation elicited by morphine (10 mg/kg, IV) in freely moving male Sprague Dawley rats was altered by prior administration of dl-PROP (25 mg/kg, IV). The major findings were 1) the effects of morphine on ventilatory parameters were not affected by subsequent injection of dl-PROP; 2) first injection of l-CYSee elicited a prompt reversal of the adverse effects of morphine that was more pronounced in dl-PROP-treated than vehicle-treated rats; and 3) the actions of the second injection of l-CYSee were dramatically augmented in dl-PROP-treated rats. In addition, the changes in many of the ventilatory parameters during a subsequent hypoxic-hypercapnic (HH) gas challenge were augmented substantially by dl-PROP. This study demonstrates that 1) inhibition of CSE with dl-PROP does not affect the ventilatory actions of morphine, 2) reversal effects of l-CYSee were augmented by blockade of CSE, and 3) blockade of CSE augments the ventilatory responses to HH gas challenge in morphine-treated rats. These unexpected findings suggest that the CSE-dependent production of H₂S from l-CYSee countermands l-CYSee reversal of morphine-induced respiratory depression in rats.NEW & NOTEWORTHY The ability of l-cysteine ethyl ester (l-CYSee) to overcome the adverse effects of morphine on breathing is exaggerated by inhibition of cystathionine-γ-lyase (CSE), suggesting that conversion of l-CYSee or l-cysteine to H₂S countermands the effects of l-CYSee against morphine.

Epithelial sodium channels (ENaCs) are essential for sodium (Na⁺) transport and maintaining fluid balance, which is vital for the removal of fetal fluid at birth and the homeostasis of luminal fluid in the lungs. In mice, ENaC is composed of three subunits (α, β, and γ). However, in humans, a fourth δ-subunit is also expressed. This study investigated the physiological role of the δ-ENaC in fetal/neonatal lungs, an area that remains less explored despite its potential significance. We measured expansion in mouse E15 lung explants expressing human δ-ENaC (SCNN1D-Tg). We found that transgenic expression of δ-ENaC enhanced fluid absorption and significantly reduced the surface area increase compared with wild-type (WT) explants (142.30 ± 5.81% vs. 163.80 ± 5.95% expansion, P < 0.001). Amiloride treatments revealed that both α-ENaC and δ-ENaC contributed to fluid absorption. No statistical significance was observed in the amiloride-sensitive fraction of SCNN1D-Tg explants compared with WT preparations in the presence of 100 µM amiloride (P = 0.400). In contrast, a significant reduction in amiloride-sensitive fraction in SCNN1D-Tg explants was observed in the presence of 10 µM amiloride (P < 0.001). Furthermore, specific blocking of α-ENaC using α-13 inhibitory peptide resulted in a 2.12-fold growth increase in WT explants, compared with a 1.47-fold increase in SCNN1D-Tg explants (P < 0.001). In summary, this study provides evidence that δ-ENaC may contribute to fluid absorption in E15 and newborn lungs, highlighting its significance in alveolar fluid regulation in prenatal and postnatal lungs.NEW & NOTEWORTHY The findings of our study highlight the significance of δ-ENaC in lung fluid regulation. Transgenic expression of human δ-ENaC contributes to fluid absorption increase, supporting its potential as a pathway for alveolar fluid clearance in E15 and postnatal lungs.

The role of the chronic obstructive pulmonary disease (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 trophic unit. HHIP expression was assessed in lung tissue and airway epithelial cells (AECs) from patients with COPD and non-COPD controls. The effect of HHIP overexpression was assessed on cigarette smoke extract (CSE)-induced changes in epithelial plasticity genes, for example, cadherin 1 (CDH1, encoding E-cadherin) in human bronchial epithelial cells (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 the lung tissue of patients with COPD, whereas the expression of HHIP was significantly lower in COPD-derived AECs compared with the 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 the 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 with 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.NEW & NOTEWORTHY We show that overexpression of HHIP protected from cigarette smoke-induced epithelial-to-mesenchymal transition and promoted epithelial regeneration via epithelial-mesenchymal cross talk in non-COPD controls. Thus, the lower expression of HHIP in airway epithelial cells from patients with COPD may contribute to abnormal epithelial repair in both proximal and distal parts of the lungs of patients with COPD.

Adaptive angiogenesis can drive repair or underlie the pathogenesis of tissue remodeling. Pulmonary vascular dysfunction is a major manifestation of chronic lung disease (CLD), but the role of angiogenesis in the development of CLD is not well defined. Microvascular capillaries in the alveolar-capillary network are the vessels most affected by pruning and remodeling in the lung, resulting in reduced capillary length and diameter with subsequent loss of gas exchange surfaces. Our lab has previously demonstrated that microvascular endothelial progenitor cells (mvEPCs) drive reparative angiogenesis. We hypothesize that visualization of the alveolar-capillary microvasculature in three-dimensions is essential to define the mechanisms governing repair versus progression to the pathogenesis of CLD. To address this gap in knowledge, we have developed a simple and reliable fluorescent perfusion technique that will allow the quantitation of microvessel structure in the alveolar-capillary network using mouse models of lung injury. This approach may be used in various organ systems to visualize microvasculature structure and its role in disease.NEW & NOTEWORTHY We developed and validated a fluorescent technology to visualize and quantify the alveolar-capillary network in three-dimensions in mouse lung for modeling of the microvasculature in models of lung disease.

Lung endothelial cells (ECs) are essential for maintaining organ function and homeostasis. Despite sharing some common features with ECs from organ systems, lung ECs exhibit significant heterogeneity in morphology, function, and gene expression. This heterogeneity is increasingly recognized as a key contributor to the development of pulmonary diseases like pulmonary hypertension (PH). In this mini-review, we explore the evolving understanding of lung EC heterogeneity, particularly through the lens of single-cell RNA sequencing (scRNA-seq) technologies. These advances have provided unprecedented insights into the diverse EC subpopulations, their specific roles, and the disturbances in their homeostatic functions that contribute to PH pathogenesis. In particular, these studies identified novel and functionally distinct cell types such as aerocytes and general capillary ECs that are critical for maintaining lung function in health and disease. In addition, multiple novel pathways and mechanisms have been identified that contribute to aberrant pulmonary vascular remodeling in PH. Emerging techniques like single-nucleus RNA sequencing and spatial transcriptomics have further pushed the field forward by discovering novel disease mediators. As research continues to leverage these advanced techniques, the field is poised to uncover novel EC subtypes and disease mechanisms, paving the way for new therapeutic targets in PH and other lung diseases.