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

The mucociliary transport apparatus is critical for maintaining lung health via the coordinated movement of cilia to clear mucus and particulates. A metachronal wave propagates across the epithelium when cilia on adjacent multiciliated cells beat slightly out of phase along the proximal-distal axis of the airways in alignment with anatomically directed mucociliary clearance. We hypothesized that metachrony optimizes mucociliary transport (MCT) and that disruptions of calcium signaling would abolish metachrony and decrease MCT. We imaged bronchi from human explants and ferret tracheae using micro-optical coherence tomography (µOCT) to evaluate airway surface liquid depth (ASL), periciliary liquid depth (PCL), cilia beat frequency (CBF), MCT, and metachrony in situ. We developed statistical models that included covariates of MCT. Ferret tracheae were treated with BAPTA-AM (chelator of intracellular Ca²⁺), lanthanum chloride (nonpermeable Ca²⁺channel competitive antagonist), and repaglinide (inhibitor of calaxin) to test calcium dependence of metachrony. We demonstrated that metachrony contributes to mucociliary transport of human and ferret airways. MCT was augmented in regions of metachrony compared with nonmetachronous regions by 48.1%, P = 0.0009 or 47.5%, P < 0.0020 in humans and ferrets, respectively. PCL and metachrony were independent contributors to MCT rate in humans; ASL, CBF, and metachrony contribute to ferret MCT rates. Metachrony can be disrupted by interference with calcium signaling including intracellular, mechanosensitive channels, and calaxin. Our results support that the presence of metachrony augments MCT in a calcium-dependent mechanism.NEW & NOTEWORTHY We developed a novel imaging-based analysis to detect coordination of ciliary motion and optimal coordination, a process called metachrony. We found that metachrony is key to the optimization of ciliary-mediated mucus transport in both ferret and human tracheal tissue. This process appears to be regulated through calcium-dependent mechanisms. This study demonstrates the capacity to measure a key feature of ciliary coordination that may be important in genetic and acquired disorders of ciliary function.

The 18-kDa isoform of basic fibroblast growth factor (bFGF/FGF2) lacks a conventional signal peptide sequence and is exported by a novel membrane-associated transport pathway. Extracellular vesicles (EVs) are increasingly recognized as mediators of intercellular communication in the lung, and our prior work demonstrates that EVs carry cargo that contributes to hyperoxic lung injury and are biomarkers for bronchopulmonary dysplasia. We used primary human bronchial epithelial (HBE), pulmonary artery endothelial (HPAE), and fibroblast (HNF) cells to determine whether FGF2 was secreted in EVs. EVs were isolated by ultracentrifugation from HBE, HPAE, and HNF exposed to either normoxia or hyperoxia, followed by nanoparticle tracking analysis and electron microscopy. Hyperoxia exposure increased the total EV number. All three cell types released FGF2-18kDa both directly into the extracellular environment (secretome), as well as in EVs. HBE released more FGF2-18kDa in EVs during hyperoxia, and these were internalized and localized to both nuclei and cytoplasm of recipient cells. By co-immunoprecipitation, we identified potential binding partners of FGF2-18kDa in the nuclei, including histone 1.2 (H1.2) binding protein, that may mediate downstream effects that do not involve FGF2 binding to cell surface receptors. FGF2-18kDa interaction with H1.2 binding protein may indicate a mechanism by which FGF2 secreted in EVs modulates cellular processes. FGF2 was also found to increase angiogenesis by Matrigel assay. Further studies are necessary to determine the biological relevance of FGF2 in EVs as modulators of lung injury and disease.NEW & NOTEWORTHY We found that multiple lung cell types release basic fibroblast growth factor (FGF2)-18kDa both directly into the extracellular environment (secretome), as well as in extracellular vesicles (EVs). Bronchial epithelial cells released more FGF2-18kDa in EVs during hyperoxia, which could be internalized rapidly by recipient cells. We also identified potential binding partners of FGF2-18kDa in nuclei that may mediate downstream effects that do not involve FGF2 binding to cell surface receptors. We also confirmed a potential angiogenic role for FGF2-18kDa.

Chitin, a polysaccharide found in the fungal cell wall and the exoskeletons of house dust mites and cockroaches, has garnered attention as a potential immunoreactive allergen. Mammals have evolved to express chitin-degrading chitinases (acidic mammalian chitinase/AMCase and chitotriosidase) that may modulate immune responses to chitin. We have previously reported that mice deficient in AMCase (Chia^-/-) demonstrated better lung function during allergic fungal asthma. As expected, we show that mice overexpressing AMCase (SPAM mice) had worse airway hyperreactivity (AHR) during allergic fungal asthma. We further demonstrate that chitin-positive Aspergillus fumigatus conidia are detectable in the allergic lung during chronic exposure. Lung function in Chia^-/- and SPAM mice is directly correlated with the level of chitinase activity during chronic fungal exposure (Chia^-/- mice, negligible chitinase activity, lower AHR; SPAM mice, heightened chitinase activity, higher AHR), suggesting that the breakdown of chitin promoted AHR. However, chronic exposure of normal mice to purified A. fumigatus chitin resulted in only moderate inflammatory changes in the lung that were not sufficient to induce AHR. Moreover, despite having dramatic differences in chitinase activity, chronic exposure of Chia^-/- and SPAM mice to purified A. fumigatus chitin likewise did not modulate AHR. Collectively, these results indicate that chronic exposure to fungal chitin alone is incapable of driving AHR. Furthermore, our data suggest that the chitinase-mediated degradation of chitin associated with A. fumigatus conidia may facilitate unmasking and/or liberation of other fungal cell wall components that drive inflammatory responses that contribute to AHR.NEW & NOTEWORTHY Humans with asthma sensitized to fungi often have more severe asthma than those who are not fungal-sensitized. Chitin makes up a significant portion of the cell wall of fungi and has been implicated as a pathogenic factor in allergic asthma. Ellis et al. demonstrate that chronic exposure to fungal chitin alone is unable to modulate lung function, even in the presence of differential lung chitinase activity.

Obesity is a risk factor for increased morbidity and mortality in viral respiratory infection. Mucociliary clearance (MCC) in the airway is the primary host defense against viral infections. However, the impact of obesity on MCC is unclear, prompting this study. Using murine tracheal tissue culture and in vitro influenza A virus (IAV) infection models, we analyzed cilia-driven flow and ciliary beat frequency (CBF) in the airway epithelium to evaluate MCC. Short-term IAV infection increased cilia-driven flow and CBF in control mice, but not in high-fat diet-induced obese mice. Basal cilia-driven flow and CBF were also lower in obese mice than in control mice. Mechanistically, the increase of extracellular adenosine triphosphate (ATP) release during IAV infection, which was observed in the control mice, was abolished in the obese mice; however, the addition of ATP increased cilia-driven flow and CBF both in control and obese mice to a similar extent. In addition, RNA sequencing and reverse transcription-polymerase chain reaction revealed the downregulation of several cilia-related genes, including Dnah1, Dnal1, Armc4, and Ttc12 (the dynein-related genes); Ulk4 (the polychaete differentiation gene); Cep164 (the ciliogenesis and intraflagellar transport gene); Rsph4a, Cfap206, and Ppil6 (the radial spoke structure and assembly gene); and Drc3(the nexin-dynein regulatory complex genes) in obese murine tracheal tissues compared with their control levels. In conclusion, our studies demonstrate that obesity attenuates MCC under basal conditions and during IAV infection by downregulating the expression of cilia-related genes and suppressing the release of extracellular ATP, thereby increasing the susceptibility and severity of IAV infection.NEW & NOTEWORTHY Our study shows that obesity impairs airway mucociliary clearance (MCC), an essential physical innate defense mechanism for viral infection. Mechanically, this is likely due to the obesity-induced downregulation of cilia-related genes and attenuation of extracellular ATP release. This study provides novel insights into the mechanisms driving the higher susceptibility and severity of viral respiratory infections in individuals with obesity.

Repair and regeneration of a diseased lung using stem cells or bioengineered tissues is an exciting therapeutic approach for a variety of lung diseases and critical illnesses. Over the past decade, increasing evidence from preclinical models suggests that mesenchymal stromal cells, which are not normally resident in the lung, can be used to modulate immune responses after injury, but there have been challenges in translating these promising findings to the clinic. In parallel, there has been a surge in bioengineering studies investigating the use of artificial and acellular lung matrices as scaffolds for three-dimensional lung or airway regeneration, with some recent attempts of transplantation in large animal models. The combination of these studies with those involving stem cells, induced pluripotent stem cell derivatives, and/or cell therapies is a promising and rapidly developing research area. These studies have been further paralleled by significant increases in our understanding of the molecular and cellular events by which endogenous lung stem and/or progenitor cells arise during lung development and participate in normal and pathological remodeling after lung injury. For the 2023 Stem Cells, Cell Therapies, and Bioengineering in Lung Biology and Diseases Conference, scientific symposia were chosen to reflect the most cutting-edge advances in these fields. Sessions focused on the integration of "omics" technologies with function, the influence of immune cells on regeneration, and the role of the extracellular matrix in regeneration. The necessity for basic science studies to enhance fundamental understanding of lung regeneration and to design innovative translational studies was reinforced throughout the conference.

Pulmonary arterial hypertension (PAH) is a progressive disease characterized by vasoconstriction and remodeling of small pulmonary arteries (PAs). Central to the remodeling process is a switch of pulmonary vascular cells to a proliferative, apoptosis-resistant phenotype. Plasminogen activator inhibitors-1 and -2 (PAI-1 and PAI-2) are the primary physiological inhibitors of urokinase-type and tissue-type plasminogen activators (uPA and tPA), but their roles in PAH are unsettled. Here, we report that: 1) PAI-1, but not PAI-2, is deficient in remodeled small PAs and in early-passage PA smooth muscle and endothelial cells (PASMCs and PAECs) from subjects with PAH compared with controls; 2) PAI-1^-/- mice spontaneously develop pulmonary vascular remodeling associated with upregulation of mTORC1 signaling, pulmonary hypertension (PH), and right ventricle (RV) hypertrophy; and 3) pharmacological inhibition of uPA in human PAH PASMCs suppresses proproliferative mTORC1 and SMAD3 signaling, restores PAI-1 levels, reduces proliferation, and induces apoptosis in vitro, and prevents the development of SU5416/hypoxia-induced PH and RV hypertrophy in vivo in mice. These data strongly suggest that downregulation of PAI-1 in small PAs promotes vascular remodeling and PH due to unopposed activation of uPA and consequent upregulation of mTOR and transforming growth factor-β (TGF-β) signaling in PASMCs, and call for further studies to determine the potential benefits of targeting the PAI-1/uPA imbalance to attenuate and/or reverse pulmonary vascular remodeling and PH.NEW & NOTEWORTHY This study identifies a novel role for the deficiency of plasminogen activator inhibitor (PAI)-1 and resultant unrestricted uPA activity in PASMC remodeling and PH in vitro and in vivo, provides novel mechanistic link from PAI-1 loss through uPA-induced Akt/mTOR and TGFβ-Smad3 upregulation to pulmonary vascular remodeling in PH, and suggests that inhibition of uPA to rebalance the uPA-PAI-1 tandem might provide a novel approach to complement current therapies used to mitigate this pulmonary vascular disease.

Development of the respiratory system can be affected by the use of drugs during pregnancy, as the prenatal phase is highly sensitive to pharmacological interventions, resulting in long-term consequences. The deleterious effects of external cannabinoids during gestation may be related to negative interference in central nervous system formation, cardiorespiratory system function, and behavioral disorders. Nevertheless, the impact of external cannabinoids on cardiorespiratory network development, chemosensitivity, and its future consequences in adulthood is still unclear. We evaluated the effects of prenatal exposure to a synthetic cannabinoid (WIN 55,212-2, 0.5 mg·kg^-1·day^-1) on the cardiorespiratory control and panic-like behavior of male and female rats in adulthood. Exogenous cannabinoid exposure during pregnancy resulted in a sex-dependent difference in breathing control. Specifically, males showed increased chemosensitivity to CO₂ and O₂, whereas females exhibited decreased sensitivity. Altered cardiovascular control was evident, with prenatally treated males and females being more susceptible to hypertension and tachycardia under adverse environmental conditions. Moreover, WIN-treated males exhibited higher fragmentation of sleep episodes, whereas females displayed anxiolytic and panicolytic behavioral responses to CO₂. However, no changes were observed in the mechanical component of the respiratory system, and there were no neuroanatomical alterations, such as changes in the expression of CB₁ receptors in the brainstem or in the quantification of catecholaminergic and serotonergic neurons. These findings highlight that external interference in cannabinoid signaling during fetal development causes sex-specific, long-lasting effects for the cardiorespiratory system and behavioral responses in adulthood.NEW & NOTEWORTHY The surge in recreational cannabis use and cannabinoid-based medication prescription among pregnant women has been notable in recent years, fueled by the misconception that natural products are inherently safe. Significant gaps persist regarding the potential risks of maternal consumption of cannabinoids and the long-term effects on the cardiorespiratory system of their offspring, which may be determined by sex. Accordingly, this research aims to diminish this lack of information and raise a note of caution.

Cross talk between T cells and airway smooth muscle (ASM) may play a role in modulating asthmatic airway inflammation and remodeling. Infiltrating T cells have been observed within the ASM bundles of asthmatics, and a wide range of direct and indirect interactions between T cells and ASM has been demonstrated using various in vitro and in vivo model systems. Contact-dependent mechanisms such as ligation and activation of cellular adhesion and costimulatory molecules, as well as the formation of lymphocyte-derived membrane conduits, facilitate the adhesion, bidirectional communication, and transfer of materials between T and ASM cells. T cell-derived cytokines, particularly of the Th1, Th2, and Th17 subsets, modulate the secretome, proliferation, and contractility of ASM cells. This review summarizes the mechanisms governing T cell-ASM cross talk in the context of asthma. Understanding the underlying mechanistic basis is important for directing future research and developing therapeutic interventions targeted toward this complex interaction.