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

The administration of opioid receptor antagonists is believed to overcome ventilatory depressant effects of opioids. Here we show that many ventilatory depressant effects of morphine are converted to excitatory responses after µ₁-opioid receptor blockade, and that these responses are accompanied by ventilatory instability. In this study, we report 1) ventilatory responses elicited by morphine (10 mg/kg, iv) and 2) ventilatory responses elicited by a subsequent hypoxic-hypercapnic (HH) gas challenge and return to room air in male Sprague Dawley rats pretreated with 1) vehicle, 2) the centrally acting selective µ₁-opioid receptor antagonist, naloxonazine (1.5 mg/kg, iv), or 3) the centrally acting (delta 1,2) δ_1,2-opioid receptor antagonist, naltrindole (1.5 mg/kg, iv). The morphine-induced decreases in frequency of breathing, peak inspiratory flow, peak expiratory flow, expiratory flow at 50% expired TV, inspiratory drive, and expiratory drive in vehicle-treated rats were converted to profound increases in naloxonazine-treated rats. Additionally, the adverse effects of morphine on expiratory delay and apneic pause were augmented in naloxonazine-treated rats, and administration of morphine increased ventilatory instability (i.e., noneupneic breathing index) in naloxonazine-treated rats, which was not due to increases in ventilatory drive. Subsequent exposure to a HH gas challenge elicited qualitatively similar responses in both groups, whereas the responses upon return to room air (e.g., frequency of breathing, inspiratory time, expiratory time, end expiratory pause, relaxation time, expiratory delay, and noneupneic breathing index) were substantially different in naloxonazine-treated versus vehicle-treated rats. The above mentioned effects of morphine were only marginally affected in naltrindole-treated rats. These novel data highlight the complicated effects that µ₁-opioid receptor antagonism exerts on the ventilatory effects of morphine.NEW & NOTEWORTHY This study shows that the systemic injection of morphine elicits a pronounced overshoot in ventilation in freely-moving Sprague Dawley rats pretreated with the centrally-acting selective µ₁-opioid receptor antagonist, naloxonazine, but not with the centrally-acting δ_1,2-opioid receptor antagonist, naltrindole. This suggests that morphine can recruit a non-µ₁-opioid receptor system that promotes breathing.

Bronchopulmonary dysplasia (BPD) is a common chronic lung disease in pediatrics. Neonatal mice placed in hyperoxia (85% oxygen, HYP) develop lung injury reminiscent of BPD. We tested the hypothesis that mice deficient in arginase-2 (Arg2KO) exposed to HYP would have attenuated lung inflammation and injury compared with similarly exposed wild-type mice. Arg2KO and C57BL/6 (WT) mice were placed in either room air (NORM) or HYP on postnatal day 0 (P0) and exposed for up to 14 days. RNAseq data on P1 and P14 showed that HYP differentially upregulated genes, particularly those related to development and inflammation, between the two genotypes. Neonatal mice exposed to HYP had evidence of alveolar simplification at P7 and P14, which was slightly attenuated in Arg2KO mice. After 14 days in HYP, mice were moved to NORM, and methacholine challenge testing was performed at 6, 8, or 12 wk of age. WT mice exposed to neonatal hyperoxia showed greater methacholine-induced respiratory system resistance (R_RS) at 6 and 8 wk of age compared with WT mice exposed to NORM. The methacholine-induced increase in R_RS in Arg2KO mice exposed to neonatal hyperoxia was not different from normoxia-exposed mice of either genotype. At 6, 8, and 12 wk, alveolar simplification was evident in both WT and Arg2KO mice exposed to neonatal hyperoxia with no differences between genotypes. These data demonstrate that Arg2KO attenuated both the hyperoxia-induced lung inflammation at P1 and P14 and the airway hyperreactivity at 6 and 8 wk of age.NEW & NOTEWORTHY Our findings suggest that inhibiting arginase 2 may be a potential therapeutic target for mitigating short-term and long-term adverse outcomes related to airway reactivity in bronchopulmonary dysplasia (BPD) that deserves further study. Furthermore, our results suggest that airway reactivity and lung architecture may be differentially regulated in neonates and may require specific and different targeting to prevent the specific outcome in neonates at risk for developing BPD.

Cystic fibrosis (CF) is a chronic disease caused by dysfunctional or absent cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is expressed in immune cells and regulates innate immunity, both directly and indirectly. The epithelial sodium channel (ENaC) contributes to dysfunction in CF airway epithelial cells. However, the impact of non-CFTR ion channel dysfunction on CF immune responses is not understood. Improved understanding of how immune function is regulated by ion channels may allow antibiotic- and mutation-agnostic treatment approaches to chronic infection and inflammation. Therefore, we hypothesized that ENaC is aberrantly expressed in CF macrophages and directly contributes to impaired phagocytic and inflammatory functions. ENaC expression was characterized in immune cells isolated from CF and non-CF blood donors. Monocyte-derived macrophage (MDM) function and bacterial killing were tested with ENaC modulation. Baseline ENaC expression in human CF MDMs, lymphocytes, and granulocytes was increased at both the transcript and protein level relative to non-CF and persisted after infection. CFTR inhibition in non-CF MDMs resulted in ENaC overexpression. CFTR modulator treatment reduced but did not eliminate ENaC overexpression in CF MDMs. Interestingly, ENaC inhibition increased CFTR expression. Amiloride-treated CF MDMs also showed normalized reactive oxygen species (ROS) production, improved autophagy, and decreased proinflammatory cytokine production. Sodium channel expression in CF MDMs normalized after amiloride treatment with minimal effect on other ion channels. In summary, ENaC modulation in immune cells is a novel potential therapeutic target for CF infection control, either in combination with CFTR modulators, or as a sole agent for people not eligible for CFTR modulators.NEW & NOTEWORTHY New research reveals that epithelial sodium channel (ENaC) overexpression in cystic fibrosis (CF) immune cells impairs macrophage function. Inhibiting ENaC increases cystic fibrosis transmembrane conductance regulator (CFTR) expression, normalizes reactive oxygen species production, improves autophagy, and reduces proinflammatory cytokine production. This suggests that ENaC modulation could be a novel therapeutic target for CF infection control, either alone or with CFTR modulators, offering new hope for patients not eligible for current treatments.

In patients with chronic obstructive pulmonary disease (COPD), lung-tissue regenerative mechanisms are thought to be exhausted, to which cellular senescence may contribute. Lung-derived mesenchymal stem/stromal cells (LMSCs) constitute a potent supportive cell type able to self-renew and promote alveolar regeneration. We hypothesized that LMSCs are less sensitive to senescence induction in COPD than other supportive cells, for example, lung fibroblasts (LFs), and therefore more promising in regenerative strategies. We compared senescence markers in LMSCs and LFs from the same subjects with/without replicative- and stress-induced senescence. LMSCs and LFs were isolated from COPD and non-COPD lung tissue using cell-specific protocols and expanded for multiple passages under the same culture conditions. Proliferation, senescence-associated β-galactosidase (SA-β-gal) activity, expression of senescence markers (CDKN2A/P16, CDKN1A/P21, and LMNB1), P21 protein levels, secretion of senescence markers (IL-6 and IL-8), and alveolar growth factors [hepatocyte growth factor (HGF) and fibroblast growth factor 10 (FGF10)] were assessed in the absence/presence of paraquat (PQ). We observed higher population doublings, and lower SA-β-gal positive cells and P21 protein levels in LMSCs compared with LFs at baseline. COPD-derived LFs had lower population doublings and higher cellular size than controls, which was not observed for COPD-derived LMSCs. LMSCs displayed lower sensitivity to PQ-induced senescence compared with LFs (COPD and control combined). Senescence induction was accompanied by increased IL-6 and IL-8 secretion, to which fibroblasts were more sensitive, and by reduced FGF10 but not HGF expression in both cell types. This study demonstrates that LMSCs have lower levels of senescence and lower sensitivity toward senescence induction compared with LFs, affecting cell expansion and FGF10 expression. This suggests that LMSCs are better suited for cell-based therapies.NEW & NOTEWORTHY We demonstrate that LMSCs are less sensitive to senescence induction by oxidative stress and replication than LFs, which was accompanied by an increased ability to expand. This makes LMSCs more suitable for cell-based therapies in COPD. As senescence affected growth factors involved in alveolar repair, specifically FGF10 expression in both LMSCs and LFs, we additionally suggest that the development of anti-senescence strategies may promote endogenous tissue repair in COPD.

It is well known that exogenous opioids such as morphine and fentanyl can depress breathing by inhibiting brainstem breathing control circuit activity. However, the role of endogenous opioids in breathing control is less clear. Endogenous opioid peptides and opioid receptors are expressed within the embryonic brainstem at the same time as when respiratory rhythm-generating neurons begin to mature. However, the extent to which endogenous opioids participate in respiratory control maturation is not known. Therefore, our goal is to review the current state of knowledge for the role of endogenous opioids in breathing control development. We set the stage by reviewing how endogenous opioid peptides regulate breathing in young and adult mammals. We describe the prenatal and postnatal development of endogenous opioid peptides and receptors in relation to breathing development. In addition, we review the effects of exogenous opioids on breathing during early life and identify areas in need of further study. We also broadly describe pain circuitry development to compare the opioid influence on nociception with how opioids impact breathing. We map the locations of endogenous opioid peptide production in the adult and developing brainstem respiratory network. Last, we propose clinical breathing conditions that may involve the endogenous opioid system. Given advances in tools for detecting endogenous opioid peptide release and the evidence reviewed herein, future research will yield new discoveries in the role of endogenous opioids in breathing across the lifespan.

Endothelial-to-mesenchymal transition (EndMT) is a biological process that converts endothelial cells to mesenchymal cells with increased proliferative and migrative abilities. EndMT has been implicated in the development of pulmonary vascular remodeling in pulmonary arterial hypertension (PAH), a fatal and progressive lung vascular disease. Transforming growth factor β₁ (TGF-β₁), an inflammatory cytokine, is known to induce EndMT in many types of endothelial cells including lung vascular endothelial cells (LVECs). An increase in cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt) is a major stimulus for cellular proliferation and phenotypic transition, but it is unknown whether Ca²⁺ signaling is involved in EndMT. In this study, we tested the hypothesis that TGF-β₁-induced EndMT in human LVEC is Ca²⁺-dependent. Treatment of LVEC with TGF-β₁ for 5-7 days resulted in increase in SNAI1/2 expression, induction of EndMT, upregulation of STIM/Orai1, and enhancement of store-operated Ca²⁺ entry (SOCE). Removal (or chelation) of extracellular or intracellular Ca²⁺ with EGTA or BAPTA-AM, respectively, abolished EndMT in response to TGF-β₁. Moreover, EGTA diminished TGF-β₁-induced increase in SNAI in a dose-dependent manner. Knockdown of either STIM1 or Orai1 was sufficient to prevent TGF-β-mediated increase in SNAI1/2 and EndMT but did not rescue the continuous adherent junctions. Blockade of Orai1 channels by AnCoA4 inhibited TGF-β-mediated EndMT and restored PECAM1-positive continuous adherent junctions. In conclusion, intracellular Ca²⁺ signaling plays a critical role in TGF-β-associated EndMT through enhanced SOCE and STIM1-Orai1 interaction. Thus, targeting Ca²⁺ signaling pathways regulating EndMT may be a novel therapeutic approach to treat PAH and other forms of precapillary pulmonary hypertension.NEW & NOTEWORTHY EndMT has been reported to contribute to the pathogenesis of PAH. In this study, we aimed to determine the role of Ca²⁺ signaling in the development of EndMT in human lung vascular endothelial cells. Our data suggest that TGF-β₁ requires store-operated Ca²⁺ entry through STIM1/Orai channels to induce SNAI-mediated EndMT. For the first time, we demonstrated that TGF-β₁-induced EndMT is a Ca²⁺-dependent event, whereas inhibition of STIM1/Orai interaction attenuated EndMT in response to TGF-β₁.

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.