Citations

Recognizing that antioxidant systems are impaired in the airways of premature infants, we used human fetal airway cells to explore the impact of oxygen on iron regulation and iron-mediated cell death (ferroptosis). We find moderate hyperoxia impairs antioxidant systems that prevent ferroptosis and dysregulates iron metabolism, while severe hyperoxia has a negative effect on mechanisms driving the early onset of ferroptosis. Inhibitors of ferroptosis decrease iron and lipid peroxidation, demonstrating links between oxygen and iron regulation in developing airways.

Abstract Rationale Premature infants exposed to supplemental oxygen are at increased risk of developing airway diseases such as bronchopulmonary dysplasia (BPD) and asthma, making it important to understand how oxygen detrimentally impacts developing airways. Even in moderation, hyperoxia promotes bronchial airway hyperresponsiveness (AHR) through effects on fetal airway smooth muscle (fASM), a cell type involved in impaired bronchodilation and remodeling. Iron dysregulation has been implicated in adult airway diseases such as asthma, COPD, and hyperoxic lung injury (HLI). Immature antioxidant systems in neonates and their increased susceptibility to iron-induced oxidative stress may lead to ferroptosis, contributing to pathologies observed in subsequent airway disease. The role of impaired iron metabolism, mediated by hyperoxia, remains largely unexplored in fetal airways. This study investigated the effects of increasing oxygen levels on antioxidant systems, iron metabolism, and lipid peroxidation in human fASM. Methods fASM from 18-20 week gestational age (canalicular stage; Mayo IRB exempt) were isolated and exposed to normoxia (21% O2) or increasing levels of hyperoxia (50%, 70%, and 90% O2) and treated with iron chelator, deferoxamine (DFO) [100uM] or reactive oxygen species (ROS) scavenger ferrostatin (Fer-1) [10uM] for 48h. Levels of intracellular iron (Fe2+) were determined using ferroOrange. Cell lysates were examined for protein expression of antioxidant markers (GPX4, SLC7A11, CBS, NRF2, KEAP1, and FSP1) and iron metabolism markers (TfR1, FTH1, and NCOA4). 4-HNE (lipid radical) expression was determined by immunofluorescence staining. Results Hyperoxia decreased SLC7A11, CBS, NRF2, KEAP1 (redox regulators), and iron storage (FTH1) at all concentrations. Iron influx (TfR1) increased under 50% and 70% O2 but not 90% O2. Cytosolic iron levels increased under hyperoxia in a dose-dependent manner and decreased with iron chelator, DFO. DFO further decreased FTH1 expression under 90% O2. 4-HNE (lipid radical) increased under 90% O2 and was reduced by ROS scavenger, Fer-1. Conclusion Our findings suggest that hyperoxia simultaneously impairs antioxidant systems and iron metabolism in fASM. Specifically, hyperoxia dose-dependently increases the labile iron pool which exhausts anti-ferroptotic mechanisms: effects which can be reduced by DFO and Fer-1. This implies the presence of a redox threshold tightly regulating iron accumulation in fASM. Overall, antioxidant impairment and iron metabolism dysfunction synergistically can contribute to pediatric airway disease: mechanisms which allow for the development of targetable strategies to blunt the impact of supplemental oxygen in the premature lung. This abstract is funded by: National Institutes of Health

Abstract Rationale Asthma in the elderly (AIE) is characterized by hyperreactive and thickened, fibrotic airways (remodeling) that are not adequately addressed by current therapies. Thus, understanding mechanisms that drive AIE becomes important. Here, airway smooth muscle (ASM) cells play important roles in normal airway physiology and asthma pathophysiology. Recent discoveries have highlighted the significance of mechanosensitive Piezo (Pz)-Piezo1 and Piezo2 in the lung. While Pz channels are highly expressed, their specific contributions to ASM function remains largely unexplored. Moreover, Pz channels play essential roles in regulating the intracellular calcium homeostasis, which further drives mitochondrial dynamics. Whether Pz signaling influences mitochondrial structure and function-a key determinant of ASM energetics and contractility is unknown and therefore of significant interest. In the present study, we hypothesize that Pz1 channel activation regulates mitochondrial structure and function via an increase in intracellular calcium in ASM, leading to metabolic changes and remodeling, characteristic of AIE. Methods Primary human ASM cells were isolated from lungs of young (<45y) and AIE (≥65y) individuals obtained post-surgery (Mayo Clinic IRB approved). Cells were exposed to mechanical stretch (mimicking breathing) in the presence of either Yoda1/Jedi2 (agonist) or GsMTx4 (inhibitor) for 24 hrs. Mitochondrial fractionation was performed, and protein expression evaluated for mitochondrial dynamics targets (MFN1, MFN2, OPA1, DRP1 and FIS1) and transcriptional markers (mt-Cox1, mt-Cox2, ATP5b and TFAM). The colocalization of mitochondria and Pz channels was observed by immunofluorescence staining. Mitochondrial morphology was examined with MitoTracker staining while function was assessed using Seahorse XF analysis. Intracellular calcium ([Ca2+]i) and mitochondrial calcium ([Ca2+]m) were measured using 5µM Fura-2/AM and 1µM Rhod-2 AM respectively with fluorescence microscopy. Results Confocal microscopy revealed that Piezo1 localizes to mitochondria in ASM. Pz1 activation promoted mitochondrial fission, evidenced by an upregulation of DRP1/FIS1 and downregulation of MFN1/OPA1, confirming a fission-dominant phenotype. Pz1 activation also increased mitochondrial branching while reducing branch length, indicating enhanced fragmentation, particularly in AIE ASM. Yoda1 treatment reduced mitochondrial oxygen consumption rates, suggesting a lower energy demand. Furthermore, Yoda1 increased both [Ca2+]i and [Ca2+]m responses to histamine (10µM), with a more pronounced effect in AIE ASM. Conclusion Overall, our findings suggest that mechanotransduction through Pz channels, particularly Pz1 regulates intracellular calcium regulation and mitochondrial structure and function in ASM, linking extracellular mechanical forces to intracellular energy homeostasis. This mechanism may contribute to the altered energetics and remodeling observed in AIE airway pathophysiology. This abstract is funded by: 24POST1241979 (NAB), R01- HL056470 (YSP), R01-HL177837 (CMP)

The studies demonstrate the differential effects of the nicotinic acetylcholine receptors (nAChRs) regulating airway hyperreactivity (AHR) and remodeling in a mixed allergen model of asthma. We compared wild-type (WT) versus global α7nAChR knockout (Glα7KO) versus smooth muscle-specific α7nAChR KO (smα7KO) mice. We found that smα7KO improved lung function and AHR in mice challenged with MA and in the presence of nicotine, whereas MA-challenged Glα7KO in the presence of nicotine showed worsened AHR.

Moderate hyperoxia (30%–60% O 2 ) in premature infants promotes bronchial airway hyperresponsiveness (AHR) via airway smooth muscle (ASM), a key regulator of bronchoconstriction, bronchodilation, and remodeling. Understanding how O 2 exposure drives long-term bronchial changes in prematurity is critical for developing therapies for airway disease across the lifespan. Premature lungs have immature antioxidant defenses, potentially due to disrupted mitochondrial dynamics, increasing susceptibility to O 2 -induced oxidative stress. Thus, mitochondrial homeostasis is highly relevant to ASM dysfunction and airway disease. We propose that hyperoxia in prematurity promotes mitochondrial dysfunction, and that the gasotransmitter hydrogen sulfide (H 2 S) mitigates O 2 -induced mitochondrial damage in developing ASM. Human fetal ASM (fASM) cells were exposed to moderate hyperoxia to investigate the effects of exogenous H 2 S donors (GYY4137, AP39) and stabilization of cystathionine β-synthase (CBS), an H 2 S biosynthetic enzyme, on mitochondrial structure and function. Hyperoxia impaired fASM cell mitochondrial integrity, while H 2 S donors in particular, or CBS stabilization attenuated adverse O 2 effects on mitochondrial morphology, ROS, respiration, calcium regulation, and contractility. These findings highlight the therapeutic potential of H 2 S in the premature lung exposed to moderate hyperoxia.

Abstract Rationale: Moderate hyperoxia (<60% O2) in premature infants promotes bronchial airway hyperresponsiveness (AHR) and impaired bronchodilation involving airway smooth muscle (ASM). Understanding mechanisms by which hyperoxia initiates long-term bronchial airway changes in prematurity is critical for novel therapies for wheezing disorders and asthma in children. Underdeveloped lungs in prematurity have immature antioxidant systems, which may be due to disrupted dynamics that promote susceptibility to oxidative stress from O2. Cytoplasmic and mitochondrial calcium signaling mechanisms and O2 metabolism are pathways highly relevant to ASM and airway disease. We propose that the novel gasotransmitter hydrogen sulfide (H2S) attenuates effects of O2 in developing ASM and hypothesize that hyperoxia in prematurity promotes cellular and mitochondrial calcium signaling dysfunction, effects which are counteracted by H2S.Methods: Human fetal (fASM) cells isolated from tracheobronchial samples of de-identified 18-22 week fetuses following fetal demise (Mayo IRB approved as exempt) were exposed to 21% vs 40% O2. fASM were treated 24-72 hours with H2S donor GYY4137, mitochondrial-specific H2S donor AP39, cystathione-β-synthase (CBS) stabilizer AdoMet, cAMP inhibitors SQ22536 or KT5720, or vehicle control (DMSO). CBS shRNA lentiviral particles were used to generate stable knockdown cell lines. Protein markers were measured by Western blot. Intracellular calcium ([Ca2+]i) was measured using 5μM Fura-2/AM and mitochondrial calcium ([Ca2+]m) with 1μM Rhod-2 AM using fluorescence microscopy. Results: CBS expression was lower in fASM exposed to 40% O2 compared to 21% O2. Lentivirus induced CBS KD was exacerbated by 40% O2. Hyperoxia increased [Ca2+]i and [Ca2+]m response to histamine (10μM) in vehicle and Scr controls compared to normoxia. CBS KD increased [Ca2+]i and [Ca2+]m responses to histamine compared to Scr fASM in 21% O2. CBS KD fASM exposed to hyperoxia showed increased [Ca2+]i and [Ca2+]m responses to histamine, but the effect was not amplified by CBS KD. [Ca2+]i and [Ca2+]m responses to histamine were decreased in normoxia by H2S donors GYY4137 (50μM) and AP39 (100nM) and CBS stabilizer AdoMet (100nM); effects which also blunted hyperoxia induced increases. SQ22536 and KT5720 inhibited effects of AP39 and GYY4137 in hyperoxia. Conclusions:Our findings suggest hyperoxia leads to [Ca2+]i and [Ca2+]m dysfunction in developing airway cells, which can be alleviated through modulation of H2S pathways, offering potential targets for future therapies to blunt hyperoxia effects on bronchoconstriction and bronchodilation. Acknowledgment: Supported by AHA Postdoctoral Fellowship 20POST35210002 (Bartman), NIH R01 HL-056470 (Prakash), and HL-160570 (Pabelick).

AbstractSupplemental O2 (hyperoxia) is a critical intervention for premature infants (<34 weeks) but consequently is associated with development of bronchial airway hyperreactivity (AHR) and asthma. Clinical practice shifted toward the use of moderate hyperoxia (<60% O2), but risk for subsequent airway disease remains. In mouse models of moderate hyperoxia, neonatal mice have increased AHR with effects on airway smooth muscle (ASM), a cell type involved in airway tone, bronchodilation, and remodeling. Understanding mechanisms by which moderate O2 during the perinatal period initiates sustained airway changes is critical to drive therapeutic advancements toward treating airway diseases. We propose that cellular clock factor BMAL1 is functionally important in developing mouse airways. In adult mice, cellular clocks target pathways highly relevant to asthma pathophysiology and Bmal1 deletion increases inflammatory response, worsens lung function, and impacts survival outcomes. Our understanding of BMAL1 in the developing lung is limited, but our previous findings show functional relevance of clocks in human fetal ASM exposed to O2. Here, we characterize Bmal1 in our established mouse neonatal hyperoxia model. Our data show that Bmal1 KO deleteriously impacts the developing lung in the context of O2 and these data highlight the importance of neonatal sex in understanding airway disease.

Rhythms in lung function and time-of-day-dependent exacerbation in disease symptoms have been noted dating back to the 5th century. Today, we recognize the 24-hour variation in normal lung function and how these fluctuations are associated with the circadian component of lung pathophysiology. Diseases of the airway such as allergic asthma, COPD, pulmonary fibrosis, pneumonia and sepsis, and lung cancer all have documented correlations with circadian rhythm and the peripheral molecular clock. Additionally, chronic exposure to particulate matter and other pollutants can induce alterations in clock genes/proteins, and disruption of the circadian system increases susceptibility to poor outcomes following insult to the airway. Investigations into targets of the molecular clock have shed light on chronotherapeutics that can be used to optimize therapeutic approaches in the clinic or leverage endogenous circadian networks to enhance efficacy of currently available treatments.

With continued smoking of tobacco products and expanded use of nicotine delivery devices worldwide, understanding the impact of smoking and vaping on respiratory health remains a major global unmet need. Although multiple studies have shown a strong association between smoking and asthma, there is a relative paucity of mechanistic understanding of how elements in cigarette smoke impact the airway. Recognizing that nicotine is a major component in both smoking and vaping products, it is critical to understand the mechanisms by which nicotine impacts airways and promotes lung diseases such as asthma. There is now increasing evidence that α7 nicotinic acetylcholine receptors (α7nAChRs) are critical players in nicotine effects on airways, but the mechanisms by which α7nAChR influences different airway cell types have not been widely explored. In this review, we highlight and integrate the current state of knowledge regarding nicotine and α7nAChR in the context of asthma and identify potential approaches to alleviate the impact of smoking and vaping on the lungs.