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

Menopause associated asthma impacts a subset of women and is less responsive to current treatments. Mechanisms driving this late-onset asthma are unknown. We recently developed a mouse model of menopause associated asthma using a combination of 4-vinylcyclohexene diepoxide (VCD) and house dust mite (HDM) exposures. The goal of this study was to determine how hormone replacement therapy during perimenopause impacts lung function and inflammation. The experimental groups included menopausal mice (VCD) with and without exposure to HDM (to model allergic airways disease) and menopausal mice with and without hormone replacement therapy (HRT; via estrogen pellet implantation). Lung function during methacholine challenge was assessed by flexiVent. Serum, bronchoalveolar lavage fluid (BALF), and histological samples were collected for assessment. Mice that received HRT during perimenopause had enhanced airway hyperresponsiveness (AHR) detected by total airway resistance (R_rs), tissue damping (G), and downward shifts in pressure-volume (PV) curves compared with controls, independent of HDM challenge. Although HRT in perimenopause resulted in decreased eosinophils in the HDM model, neutrophil levels and mucus production were unchanged. Mice receiving HRT in perimenopause also had significantly increased collagen production and inflammation associated with large and small airways, independent of HDM challenge. HRT given during perimenopause may be detrimental to lung responses, including increased AHR and decreased lung function, as well as increased tissue inflammation and airway remodeling.NEW & NOTEWORTHY Menopause-associated asthma is a subtype of asthma that is still largely unexplored and difficult to manage. Women experiencing menopause-associated asthma often have more severe exacerbations, higher rates of exacerbation, and most importantly, poor response to standard treatments. This study examined the impact of estrogen replacement given during the perimenopause phase on lung inflammation and function after menopause. While decreasing eosinophil recruitment, estrogen replacement actually led to worse lung function and more airway remodeling.

Pulmonary hypertension (PH) is a progressive vascular disease driven by pulmonary arterial remodeling, characterized by cellular hyperproliferation, resistance to apoptosis, and phenotypic plasticity. Our laboratory has shown that the proton-gated cation channel, acid-sensing ion channel 1a (ASIC1a), is essential for the development of chronic hypoxia (CH)-induced PH in rodents. Importantly, ASIC1a activation occurs without changes in total ASIC1a levels but reflects a hypoxia-dependent redistribution to the plasma membrane in pulmonary arterial smooth muscle cells (PASMCs). In neurons, mitochondrial-localized ASIC1a (mtASIC1a) contributes to oxidative stress-induced mitochondrial membrane potential (ΔΨm) depolarization and apoptosis. Although mtASIC1a has not been described in vascular cells, its role in PASMCs may be relevant to mitochondrial dysfunction and apoptosis resistance in PH. We hypothesize that mtASIC1a is a crucial regulator of PASMC mitochondrial homeostasis, and its loss following CH promotes mitochondrial dysfunction and apoptosis resistance. Consistent with this, mtASIC1a localization was decreased in PASMCs and intrapulmonary arteries from CH rats compared with controls. Functionally, PASMCs from CH rats or Asic1a knockout mice exhibited ΔΨm hyperpolarization, elevated mitochondrial Ca²⁺ and superoxide, impaired mitophagy, and reduced cleaved caspase-3. Transmission electron microscopy revealed mitochondrial morphological changes, including increased size and circularity, decreased aspect ratio, and reduced mitochondrial number per cell, whereas fusion/fission proteins remained largely unchanged. Lentiviral restoration of mtASIC1a prevented ΔΨm hyperpolarization and restored caspase-3 cleavage. These findings identify mtASIC1a as a novel regulator of mitochondrial function in PASMCs, where its loss following CH promotes ΔΨm hyperpolarization, impaired mitophagy, and resistance to apoptosis.NEW & NOTEWORTHY This study identifies mitochondrial acid-sensing ion channel 1a (mtASIC1a) as a novel regulator of mitochondrial homeostasis in pulmonary arterial smooth muscle cells (PASMCs). Critically, mtASIC1a deficiency in PASMCs following in vivo chronic hypoxia or genetic deletion promotes mitochondrial membrane potential (ΔΨm) hyperpolarization, Ca²⁺ and superoxide (O₂^-) accumulation, impaired mitophagy, and caspase inhibition. Restoring mtASIC1a by lentiviral transduction prevents ΔΨm hyperpolarization and restores caspase cleavage, highlighting its importance in mitochondrial signaling and hypoxic pulmonary hypertension pathophysiology.

Obesity is a risk factor for increased prevalence and severity of asthma, particularly in females. As adults, females have an increased prevalence of asthma compared with males. Yet, the mechanisms remain unclear on how sex hormones and obesity increase airway inflammation. We hypothesize that estrogen signaling through estrogen receptor-alpha (ER-α) in T cells increased airway inflammation in the context of obesity. To test our hypothesis, we utilized a high-fat diet on female and male mice that underwent ovariectomy or gonadectomy or in Esr1^fl/fl X Cd4^Cre+ male and female mice. As controls, mice in similar groups were fed normal chow. After 8-12 wk on diets, house dust mite sensitization and challenge occurred in all mice. Lungs and bronchoalveolar lavage fluid were harvested 24 h after the last challenge. Ovarian hormones and ER-α signaling in T cells increased eosinophils, neutrophils, and Th17-mediated airway inflammation in the lungs of obese female mice. In addition, using peripheral blood mononuclear cells (PBMCs) from a well-characterized cohort of asthmatic participants, we determined that women with asthma and obesity had increased Th17 cells compared with men with asthma and obesity. Our results show that ER-α signaling in T cells increases Th17-mediated airway inflammation in obese mice and that Th17 cells circulate at higher frequencies in women with asthma compared with men with asthma. Further research into the interplay between hormonal signaling and immune responses in asthma is essential for developing personalized treatments.NEW & NOTEWORTHY Estrogen receptor-alpha (ER-α) signaling increased obesity and allergen-induced airway inflammation in mice. In addition, women with obesity and asthma had increased circulating Th17 cells compared with men with obesity and asthma. These findings provide mechanistic insights into the intersection of obesity, sex hormones, and airway inflammation-underscoring the importance of personalized approaches to managing individuals with obesity and asthma.

Pulmonary vascular remodeling contributes to persistent pulmonary hypertension of the newborn (PPHN); the mechanisms remain unknown. 5'-AMP-activated protein kinase (AMPK) is a critical regulator of energy balance and metabolism. We investigated the hypothesis that decreased AMPK function in pulmonary artery smooth muscle cells (PASMCs) leads to impaired mitochondrial capacity to perform oxidative phosphorylation and altered notch ligand expression, which together promote vascular remodeling in PPHN. Studies were performed in fetal lambs with PPHN induced by prenatal ductus arteriosus constriction and gestation-matched controls. For in vitro studies, PPHN PASMCs were treated with AMPK agonists, A769662 or metformin, and compared with untreated control and PPHN PASMCs. Expression of phosphorylated-AMPK (p-AMPK) and its downstream mediators, peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α), mitochondrial electron transport chain (ETC) complexes, differentiation markers, and notch ligands were assessed using immunoblotting in control and PPHN PASMCs. For in vivo studies, PPHN lambs were treated with metformin and were compared with untreated control and PPHN lambs. Lung sections from in vivo experiments were evaluated through immunofluorescence. Compared with controls, p-AMPK, PGC-1α, and ETC complexes were decreased in PPHN PASMCs and lung sections. PASMC differentiation marker, myosin heavy chain 11, was reduced in PPHN lung sections, whereas dedifferentiation marker, vimentin, was increased. Expression of Jag1 and Hey1 in Notch pathway was reduced in PPHN PASMCs and lung sections. A769662 and metformin increased the expression of PGC-1α, ETC complexes I and IV, Jag1, and Hey1 in PPHN PASMCs. Decreased AMPK function contributes to reduced mitochondrial oxidative phosphorylation capacity, less differentiated PASMCs, and imbalanced notch signaling, promoting remodeling in PPHN.NEW & NOTEWORTHY Our study reveals a novel mechanism for pulmonary vascular remodeling in persistent pulmonary hypertension of the newborn (PPHN). We identify a decrease in the function of a key energy sensor, AMPK, as contributing to pulmonary vascular remodeling through decreased mitochondrial oxidative phosphorylation capacity, altered differentiation marker expression, and notch ligand imbalance. Our studies may provide translational significance as restoring AMPK function offers a new therapeutic target in PPHN to improve postnatal transition in PPHN.

Neonatal airway development and injury are poorly understood, in part due to challenges of studying extremes of phenotype in human pathological samples and difficulties obtaining relevant comparator samples. Ex vivo model systems are needed to improve understanding of airway development, injury, and repair in the neonatal lung. We optimized a protocol for organotypic culture of primary murine neonatal tracheal epithelial cells (MNTECs). We compared expansion and differentiation properties of MNTECs in five different media conditions, ranging from previously published "lab-made" media to commercial sources of media. We measured the success of our organotypic cultures by quantifying the relative proportions of ciliated epithelium, TP63+ basal stem cells, and stromal cell contamination, as well as total cell numbers and air-liquid interface (ALI) thickness. Commercially available media performed better than standard lab-made media, with nearly 100% success and 20% success, respectively. Proliferation in commercial media improves expansion of TP63+ basal cells, inhibits growth of contaminating stromal cells, and improves differentiation to a polarized, ciliated pseudostratified airway epithelium, when compared with lab-made LP media. These results provide a reliable technique for studying neonatal airway epithelial cells in wild-type and genetically mutant mice.NEW & NOTEWORTHY In this study, we optimized murine neonatal tracheal epithelial cell (MNTEC) growth and differentiation for mechanistic studies of disrupted airway epithelial cell development, injury, and repair. Our protocol removes significant complexity and historical variability of murine air-liquid interface (ALI) cultures and is specifically designed for neonatal airway epithelial cell cultures with limited cell numbers. We also compared the transcriptomes of well-differentiated and poorly differentiated organotypic airway epithelial cultures to identify key genes for epithelial growth and polarization.

Obesity, a key risk factor for severe asthma, is associated with worsening symptoms and poor responses to conventional therapies. Recent studies have highlighted the presence of adipocytes within airway walls, which correlates positively with body mass index (BMI). However, the role of adipocytes in asthma pathogenesis remains largely unknown. This study aims to explore their potential contribution to airway fibrosis, a progressive form of the disease, through fibroblast-to-myofibroblast transition (FMT). In vitro coculture models were developed to investigate the interactions between adipocytes (derived from patients with and without obesity) and fibroblasts (from patients with and without asthma) on FMT. Proteomic and multiplex analyses were used to identify potential mediators of adipocyte-induced FMT. Our data revealed a significant increase in fibrogenic markers, such as alpha-smooth muscle actin and vimentin, in fibroblasts cocultured with obese (Ob) adipocytes. Notably, this transition was more pronounced in asthmatic fibroblasts compared with healthy fibroblasts. Proteomic profiling of cocultured Ob-adipocytes and asthmatic fibroblasts identified several significantly upregulated proteins linked to the regulation of the transforming growth factor-beta (TGF-β) signaling pathway, including inhibin A, latent TGF-β binding protein 1, thrombospondin 1, and follistatin. The role of TGF-β was further substantiated by multiplex assays, which demonstrated a significant increase in TGF-β and leptin production by Ob-adipocytes following coculture. These findings suggest that Ob-adipocytes may promote FMT in fibroblasts, especially asthmatic fibroblasts, by activating the TGF-β signaling pathway. This highlights a potential mechanism by which obesity exacerbates asthma severity and fibrosis, providing new avenues for therapeutic intervention.NEW & NOTEWORTHY Adipocytes have been found in the airway wall of patients with obesity. This study is the first to show that adipocytes derived from patients with obesity can induce features of airway remodeling that is seen in patients with asthma such as fibroblast-to-myofibroblast transition via the TGF-beta signaling pathway in an indirect mode of cellular communication. This highlights a potential mechanism by which obesity exacerbates asthma severity and fibrosis, providing new avenues for therapeutic intervention.

Lung implantable devices, such as stents and valves, are used as treatment for lung cancer and chronic obstructive pulmonary disease (COPD). They apply continuous compressive stress to airway tissue, potentially triggering adverse effects such as chronic inflammation, granulation tissue hyperplasia, and fibrosis at the implant site. To identify the biological responses underlying this process, we developed an in vitro contact-compression model that applies variable compressive stress to bronchial epithelial cells. Confluent layers of bronchial epithelial cells (16HBE) were subjected to compressive stress using agarose-embedded weights (3, 6, 9, and 15 g). After 24 h, cell viability, inflammation, fibrosis, and mechano-transduction were assessed using cell viability assays, quantitative real-time PCR, ELISA, and immunofluorescent staining. Maximum compressive stress (15 g) led to reduced cell viability. Compression increased the expression of inflammation, CXCL8, TNF, IL1α, GM-CSF, and remodeling-related genes, EGR1, TNC, COL1A1, and CTGF, whereas no changes in TGFB1, TNC, and FN1 expression were observed. These changes were reflected in protein levels with increased CXCL8, IL-1α, and connective tissue growth factor (CTGF) in supernatant upon compression. Compressed cells showed increased actin polymerization, mechanoreceptor relocalization, and Yes-associated protein (YAP) nuclear translocation, reflecting a mechanotransducive response. We developed a viable in vitro model to study contact-compression, showing biomechanical inflammatory and remodeling responses. With adjustable components, this model can be applied to further study tissue responses to lung implants.NEW & NOTEWORTHY Our research introduces a novel in vitro model to study how contact-compressive stress drives pathological wound-healing responses in bronchial epithelial cells. By linking mechanical loading to mechanosensory-redistribution, cytoskeletal remodeling, and both pro-inflammatory (CXCL8, IL6, IL1A, and GM-CSF) and profibrotic gene expression (CTGF, COL1A1, and EGR1), this work provides critical insights into the cellular mechanisms underlying lung implant-associated complications and offers a platform for future biomaterial and device testing.

Chronic obstructive pulmonary disease (COPD) is the third leading cause of death globally, with progressive emphysema driven by repeated epithelial damage and impaired repair. Recently, we found that secretion of cytokine receptor agonist of nuclear factor κB signaling ligand (RANKL) is higher from lung fibroblasts of patients with COPD compared with control and that RANKL reduces lung epithelial cell death. However, the underlying mechanisms, their conservation across species, and the specific epithelial cell types involved remain unclear. To investigate how RANKL affects lung epithelial cells, we used in vitro and in vivo models. Primary lung organoids from human and mouse epithelial cells assessed progenitor activation and expansion. Furthermore, we used a murine model of elastase-induced alveolar injury to examine which epithelial cell types were affected by RANKL in vivo and whether this was altered upon lung damage. RANKL supplementation increased alveolar organoid formation in both murine and human cultures compared with vehicle-treated controls. In elastase-treated mice, RANKL administration during the early repair phase enhanced the proportion of EpCAM⁺ epithelial cells and increased transitional epithelial cell states characterized by keratin 8 (Krt8) and major histocompatibility complex II (MHCII) expression following elastase-induced injury. RANKL signaling promoted epithelial regeneration by expanding alveolar progenitor and transitional epithelial cell populations, with consistent effects across human and murine models. These findings identify RANKL as a novel modulator of epithelial repair and suggest that controlled activation of this pathway could enhance lung regeneration in diseases such as COPD.NEW & NOTEWORTHY This study describes for the first time a role for bone cytokine RANKL in lung epithelial repair following lung damage. Using both murine and human models, we describe how RANKL-mediated signaling promotes epithelial expansion by acting on epithelial transitional cell states. For diseases with substantial lung damage, modulating RANKL signaling during the early repair phase following lung injury could represent a strategy to enhance epithelial regeneration.

Asthma is a chronic respiratory condition influenced by genetic, environmental, and sex-related factors. Women experience greater asthma severity, airway hyperresponsiveness (AHR), and inflammation than men, likely due to sex-linked genetic and hormonal differences. However, the independent contributions of sex chromosomes and gonadal sex to these responses remain unclear. This study examines their roles in allergic airway responses using the four core genotype (FCG) mouse model, which distinguishes between chromosomal and gonadal influences. We hypothesized that XX-mice and those with female gonads would exhibit heightened airway inflammation and immune activation in response to house dust mite (HDM) challenge. Using a controlled, moderate 5-wk HDM exposure paradigm that reliably induced allergic airway inflammation, we aimed to capture biologically relevant sex- and genotype-dependent variations rather than a maximal inflammatory phenotype. FCG mice (XXF, XXM, XYF, and XYM) underwent 5 wk of HDM exposure, followed by assessments of airway lung function and inflammation. Our results showed that HDM challenge differentially increased airway resistance and elastance in FCG mice, with specific contributions of sex chromosomes and gonadal sex. Histological analysis showed higher lung inflammation and goblet cell hyperplasia in challenged mice with female gonads than in those with male gonads. Flow cytometry assessment revealed elevated eosinophils in XXF mice. Combined, our findings show that both sex chromosomes and gonadal sex influence airway inflammation and immune responses to allergen challenge, with mice bearing XX chromosomes and female gonads exhibiting greater susceptibility.NEW & NOTEWORTHY This study provides new insights into how sex chromosomes and gonadal sex independently and interactively shape immune cell responses during allergic airway inflammation. Using the four core genotype (FCG) mouse model, we show that both genetic and hormonal factors significantly influence pulmonary immune cell populations after allergen exposure. These findings advance our understanding of the biological basis for sex differences in asthma and highlight the need for sex-informed approaches in respiratory disease research and therapy development.