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Over 37% of pregnant women report excessive daytime sleepiness or hypersomnia during the first trimester, and commonly throughout the remaining trimesters of pregnancy and the postpartum period. The clinical presentation and symptoms of central disorders of hypersomnolence (CDH) such as narcolepsy, idiopathic hypersomnia, or excessive daytime sleepiness secondary to shift work or obstructive sleep apnea may be confounded by physiological changes in sleep architecture and hormonal fluctuations occurring during pregnancy. These overlapping features may cause delays in diagnosis and treatment, impacting quality of life, maternal functioning, and overall health. Management of CDH during pregnancy and lactation requires careful consideration of maternal benefits, potential side effects of medications and fetal and neonatal safety. Here, we present a case of idiopathic hypersomnia and detail management and outcomes during pregnancy and lactation. We briefly review the existing literature on prevalence, pathophysiological mechanisms, diagnostic challenges, objective findings, and risks posed by CDH in pregnancy and lactation. In this narrative review for primary care clinicians, we also summarize currently available evidence regarding the efficacy, teratogenic and other risks of pharmacologic agents used in the treatment of CDH in pregnancy and lactation. Lastly, we provide a clinical algorithm for management of CDH during pregnancy and lactation.

Abstract Endothelial-to-mesenchymal transition (EndMT) is a biological process through which lung vascular endothelial cells (ECs) transdifferentiate into mesenchymal-like cells. EndMT has recently been implicated in the development and progression of pulmonary vascular remodeling in pulmonary hypertension (PH); however, its underlying regulatory mechanisms remain incompletely understood. MicroRNAs (miRNAs) are key post-transcriptional regulators of EC gene expression and cellular responses to various stimuli. Notably, microRNA-153 (miR-153) has been shown to directly target SNAI1 to modulate epithelial-to-mesenchymal transition (EMT), a process closely related to EndMT and extensively studied in cancer. Whether miR-153 also participates in EndMT regulation, however, remains unknown. In this study, we demonstrate that 72-hour hypoxic exposure induces SNAI1-mediated EndMT in human lung vascular ECs. Hypoxia also increased cell proliferation and disrupted intercellular junctions, leading to enhanced endothelial permeability. Reduced miR-153 expression was observed in both hypoxia- and TGF-β1-induced EndMT, as well as in ECs isolated from PH patients exhibiting an EndMT phenotype. Similar to hypoxia, TGF-β1 promoted EC permeability. Loss of miR-153 enhanced SNAI1-mediated EndMT, endothelial survival, and permeability under normoxic conditions, whereas miR-153 overexpression attenuated EndMT induced by hypoxia or TGF-β1. However, miR-153 restoration did not completely restore endothelial barrier integrity disrupted by these stimuli. In vitro findings were validated in experimental PH model. In conclusion, miR-153 serves as a critical regulator of EndMT, maintaining endothelial identity and barrier function. Therapeutic delivery of miR-153 may therefore represent a novel strategy to inhibit EndMT and attenuate pulmonary vascular remodeling in PH.

Abstract Rationale Premature birth necessitates interventions such as supplemental O2 (hyperoxia, typically <60% O2) with or without respiratory support primarily using nasal CPAP. The static stretch of CPAP can induce sustained airway hyperresponsiveness (AHR) and airway thickening (remodeling) even after CPAP is stopped - effects that contribute to a wheezing phenotype and predispose to asthma, major sources of respiratory morbidity in former “preemies.” Effects of CPAP can impact airway smooth muscle (ASM), a key cell type in asthma. We sought to find out if mechanosensitive Piezo (PZ) channels in developing ASM contribute to CPAP-induced AHR and remodeling. In other cell systems, PZ affects the Wnt/β-catenin (β-cat) pathway, but there is currently little information on how stretch influences this pathway and its downstream role in developing ASM. Our overall hypothesis is that in developing airways, PZ channels and downstream Wnt/β-cat pathway are linked in the context of stretch effects, leading to AHR and remodeling. Methods Fetal ASM (fASM) cells were isolated from lungs of 18-20-week fetuses (Mayo IRB exempt). Neonatal mice (comparable to late gestation humans) were also used; lung tissue was harvested following IACUC protocols. Western blot (WB), qRT-PCR, immunofluorescence imaging (IF), extracellular matrix (ECM) deposition assay, proliferation assay, and precision cut lung slice (PCLS) imaging were performed. Stretch conditions used were “non-stretch” (0% static strain imposed on 5% cyclic strain representing normal breathing) or “stretch” (5-10% static strain imposed on 5% cyclic strain, representing superimposed CPAP). Compounds used were Yoda1 (PZ1 agonist), GsMTx4 (PZ inhibitor), DKK1 (Wnt pathway inhibitor), and Wnt3a ligand. Results PZ1 and PZ2 are expressed in fASM and stretch increased expression of both, shown by qRT-PCR and WB. In neonatal mice exposed to CPAP, lumen area of PCLS were decreased compared to control; GsMTx4 alleviated this effect. Stretch increased ECM in fASM, however the effect was blunted by GsMTx4. Stretch increased mRNA for canonical Wnt pathway elements and decreased negative regulatory elements. Stretch increased GSK3β protein phosphorylation and β-cat protein nuclear translocation; effects blunted by DKK1. Wnt3a ligand increased proliferation and ECM in fASM. Stretch increased Wnt3a mRNA and β-cat protein nuclear translocation, with effects blunted by GsMTx4. Stretch alone or in combination with Wnt3a ligand, as well as Yoda1 alone, increased nuclear β-cat, shown qualitatively by IF. Conclusions Our study showed that Wnt/β-cat may be a novel, important, and conceivably targetable pathway (e.g. with PZ modulators) in alleviating potentially long-term, detrimental airway responses to CPAP. This abstract is funded by: NIH R01 HL056470 (Prakash), R01 HL177837 (Pabelick)

Abstract Rationale Respiratory disease is a major cause of mortality in Down syndrome (DS; trisomy 21, T21). DS is considered a progeroid syndrome, with elevated cellular senescence (SEN) and oxidative stress. Because SEN shapes organogenesis, its dysregulation may impair lung morphogenesis. We previously found SEN/SASP (senescence-associated secretory phenotype) abnormalities in prenatal human T21 lungs. Here, we test whether these alterations persist and change after birth. Methods We analyzed human lung single-nuclear RNA (snRNA-seq) datasets (prenatal and postnatal) to quantify epithelial and mesenchymal SEN/SASP signatures. Sex- and age-matched T21 and non-T21 prenatal and postnatal lungs were studied; fibroblasts were isolated as prenatal human lung fibroblasts (HFLF) and postnatal human lung fibroblasts (HLF). SEN/SASP were measured by RT-qPCR and immunofluorescence (IF) in tissue and HFLF/HLF. HFLF/HLF were assayed for SA-β-gal, oxidative stress (CellROX/MitoSOX), and Ca²+ dynamics (Fura-2 AM). 48h treatments with tunicamycin (1µM, SEN inducer), 4-phenylbutyrate (4-PBA, 2 mM, ER Stress inhibitor), or pyrroloquinoline quinone (PQQ, 20 μM, antioxidant) were performed on T21 and non-T21 HFLF/HLF. Conditioned media (CM) were collected after 48h from untreated cultures and then applied to cells of the opposite genotype for 24, 48, and 72h. Results Overall SEN profile was similar in epithelium and mesenchyme across ages, but pathway signatures differed. Epithelium shows an inflammatory SASP, while mesenchyme shows a less-inflammatory, remodeling/growth-factor SASP. These lineage patterns were more evident postnatally. Whole-tissue IF showed increased γ-H2AX and p21 in T21 vs non-T21 both prenatally and postnatally (p < 0.05). At baseline, T21 HFLF/HLF had higher CDKN1A, CDKN2A, TP53, CDKN2B, and SERPINE1 (p < 0.05; n = 5-6), with increased γ-H2AX/p21 IF (p < 0.01; n = 6), SA-β-gal (p < 0.05; n = 5-6), and ER/mitochondrial stress (p < 0.05; n = 5-6). Fura-2 AM showed greater Ca²+ release in T21 HFLF/HLF at baseline and after acetylcholine (10 μM, p < 0.01; n = 5). Mitochondrial-morphology genes (FIS1, DNM1L, MFN1) were elevated in T21 HFLF/HLF (p < 0.05; n = 6). In T21 HFLF, 4-PBA and PQQ each reduced SA-β-gal (p < 0.01; n = 6). In postnatal HLF, PQQ had the stronger effect (p < 0.05; n = 5). T21 CM induced progressively greater SEN/SASP over 24/48/72h in non-T21 cells (p < 0.05; n = 5-6), while non-T21 CM partially rescued T21-HLF phenotypes at 24h only (p < 0.05; n = 5-6). Conclusion These data indicate that lung senescence in T21 initiates in utero and intensifies after birth and reflects lineage-specific pathways. ER-stress-related mechanisms are more prominent prenatally, while mitochondrial/redox mechanisms are predominant postnatally. This evidence suggests age and lineage-dependent drivers of senescence that may help explain DS lung disease and highlight potential new mechanistic targets. This abstract is funded by: NIH/NHLBI

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)

Abstract Rationale Pulmonary Fibrosis (PF) is a fibroproliferative disease characterized by increased proliferation, constant extracellular matrix (ECM) deposition, and dysfunctional antioxidant systems. In PF, an emerging initiator is abnormal iron regulation and lipid peroxidation: consequences of iron catalyzed cell death, i.e. ferroptosis (FP). Abnormal iron regulation and lipid peroxidation lead to diminished glutathione (GSH)/glutathione peroxidase 4 (GPX4) antioxidant systems which promote ECM signaling in fibroblasts. Accordingly, inhibiting ferroptosis and/or correcting iron dysregulation via increasing GSH can help with early PF intervention. We hypothesize that enhanced hydrogen sulfide (H2S) signaling through exogenous H2S donors GYY4137 (slow-releasing) and AP39 (mitochondrial-specific), can blunt FP/lipid peroxidation leading to reduced ECM deposition in the context of PF. Methodsand Results Human fibroblasts were isolated from lung tissue of patients with PF and healthy patients (Mayo IRB approved) and were treated with exogenous H2S donors. Prussian blue staining of FFPE lung sections from patients with PF revealed increased ferric iron in alveolar regions. Immunostaining of PF human lung tissue showed lower expression of H2S generating enzyme, CBS, and higher expression of the lipid peroxidation enzyme, ACSL4, compared to non-PF patients. At the cellular level, Western blot was performed in fibroblasts to understand the effect of PF on Fe regulation. PF fibroblasts showed higher expression of transferrin receptor (TfR1) and ferritin, as well as higher expression of ACSL4. The impact of exogenous H2S donors on PF fibroblasts was tested. While H2S donors did not impact TfR1 or ferritin, they did blunt lipid peroxidation as determined using live cell BDP 581/591 C11- fluorescent imaging and ACSL4 protein expression. Furthermore, GYY4137 increased the cysteine transport protein xCT(SLC7A11; part of xC), the H2S synthesis enzyme CBS and GPX4, overall showing an antioxidant effect. This was further substantiated though measuring GSH/GSSG ratios via luminescence assay. In PF fibroblasts, GSH/GSSG levels were low comparatively to normal fibroblasts. However, with exogenous H2S, PF fibroblasts showed increased GSH/GSSG levels indicating a restoration of antioxidant function. Deposition of collagen-1 was higher in PF fibroblasts, and was blunted by GYY4137, which was more potent than the FP inhibitor ferrostatain-1. Conclusion Our data suggests ferroptosis/impaired antioxidant systems are significant factors in PF and that exogeneous H2S can be leveraged to replenish antioxidant systems and blunt FP, thus potentially reducing fibrotic activity. This abstract is funded by: NIH T32 HL 176414 (Ravi), R01 HL160570 (Pabelick, Prakash), R01 HL158532 (Prakash)

Abstract Rationale Bronchopulmonary dysplasia (BPD) is a chronic lung disease that significantly affects premature infants, leading to impaired lung development, inflammation, and fibrosis due to sustained exposure to high oxygen levels in the Neonatal Intensive Care Unit (NICU). In BPD, airway narrowing and hyperreactivity are common, making bronchodilation beneficial. Caffeine, a respiratory stimulant typically used for apnea of prematurity, also has bronchodilatory properties. This study investigates caffeine’s impact on airway remodeling (fibrosis and cellular proliferation) in fetal airway smooth muscle cells (fASMs) exposed to mild hyperoxic stress. By inhibiting phosphodiesterase, caffeine increases cyclic adenosine monophosphate (cAMP) levels, thereby relaxing airway smooth muscle. Caffeine’s influence on calcium (Ca2+) kinetics is essential for muscle contraction and relaxation. The goal is to better understand caffeine’s therapeutic role in bronchodilation and reducing fibrosis and proliferation in BPD. Methods Ca2+ imaging, proliferation assay, and immunofluorescence imaging (IF) were performed. fASMs were exposed to normoxia (21% O2) or mild hyperoxia (∼40% O2) for 48 hours, with or without caffeine treatment. Results Acute caffeine exposure (25 mM) decreased Ca2+ amplitude (corresponding to a dilatory effect) after addition of acetylcholine (10 μM) in normoxia. Caffeine treatment reduced cell proliferation in both normoxic and hyperoxic groups. Caffeine decreased both Ki67 (marker of proliferation) and Col1a1 (marker of fibrosis) protein expression under hyperoxic conditions. Conclusions Caffeine influences Ca2+ signaling, reduces cell proliferation and fibrosis, and, thus, may modulate airway remodeling and hyperreactivity under hyperoxic stress, suggesting its potential as a therapeutic strategy for BPD. This abstract is funded by: NIH R01 HL56470, R01 HL177837

Abstract Rationale Lung fibroblasts (FBs) play a pivotal role in airway remodeling in asthma. During profibrotic and inflammatory conditions, FB undergoes myofibroblast differentiation, which contributes to airway wall thickening, stiffness, and airflow obstruction. Myofibroblast differentiation is a process in which fibroblasts transform into highly contractile fibers in response to profibrotic mediators such as transforming growth factor-beta 1 (TGF-β1). This transition is characterized by increased production of alpha-smooth muscle actin (α-SMA), and the formation of actin stress fibers, which are crucial for contractile machinery. Our previous findings identified the important role of Kisspeptin/KISS1R (Kp/KISS1R) in airway smooth muscle remodeling. However, there is no information about the expression and function of Kp/KISS1R in the lung FB. This study investigates the expression of Kp/KISS1R in lung FB and their role in myofibroblast transition. Methods Primary human lung FBs were isolated from surgical lung resections (Mayo IRB-approved) and cultured in DMEM-high glucose medium with 10% fetal bovine serum and 1% antibiotic/antimycotic. Upon reaching ∼90% confluence, cells were serum-deprived and treated with kisspeptin-10 (Kp-10, 1 µM) in the presence or absence of TGF-β1 (10 ng/mL). Expression of Kisspeptin, KISS1R and α-SMA in FB cells were assessed by western blotting and confocal microscopy. FB filamentous (F)-actin formation was analyzed by staining with phalloidin. In addition, collagen gel contraction assays were performed to study fibroblast contractility. Results We found the ubiquitous expression of kisspeptin and KISS1R in human FB. Notably, we observed that KISS1R expression was significantly reduced in asthmatic FB compared to non-asthmatic FB cells. Consistent with this observation, exposure of profibrotic mediator TGF-β1 effectively reduced the expression of KISS1R in non-asthmatic FB. Furthermore, TGF-β1 exposure significantly increased the α-SMA expression in human FB cells, a myofibroblast marker. Treatment with Kp-10 significantly reduced TGF-β1-induced α-SMA expression in FB cells. TGF-β1 stimulation in human FB increased the F-actin formation. Kp-10 significantly attenuated TGF-β1-induced F-actin formation in FB cells. Additionally, chronic TGF-β1 exposure enhanced collagen gel contraction by FBs, while Kp-10 treatment effectively reduced this contraction. Conclusion This study demonstrates that activation of Kp/KISS1R signaling attenuates TGF-β1-induced myofibroblast differentiation in lung FB, suggesting KISS1R as a potential therapeutic target against airway remodeling. This abstract is funded by: NIH grant R01-HL146705 (Venkatachalem)

Abstract Rationale Bronchial airways are exposed to continual mechanical forces such as normal breathing, sighs, and clinically applied pressures such as CPAP that influence airway smooth muscle mechanics. Asthmatic airways experience exaggerated mechanical stress due to bronchoconstriction and airway wall thickening (remodeling) which contributes to lung injury. Mechanical stress is known to alter redox balance and ion channel signaling, yet its impact on iron homeostasis in asthmatic human airway smooth muscle cells (hASMs) remain unclear. Ferroptosis and iron dysregulation have emerged as significant factors in epithelial damage and airway inflammation in asthmatic models. In addition, recently discovered mechanosensitive Piezo (PZ) channels play a critical role in calcium influx upon mechanical stimulation and have been shown to contribute to iron dysregulation. In this study, we used hASMs to test the hypothesis that asthmatic patients experience iron dysregulation in the presence of mechanical stimulation. Methods hASMs and lung tissues from adult male and female patients with/without asthma were used (approved by Mayo IRB). Cells were pretreated with either PZ inhibitor GsMTx4 [1 µM] or PZ1 agonist Yoda1 [10 µM] 45 minutes before stretch. Cells were exposed to normal breathing stretch conditions on a FlexCell System at a 5% cyclic strain (15 cycles/minute) or 10% static stretch superimposed onto normal breathing for 24 hours. Cell lysates were examined for iron accumulation and antioxidant and lipid peroxidation markers (FTH1, TfR1, GPX4, and SLC7A11). Levels of intracellular ferrous iron (Fe2+) were determined using the fluorescent dye FerroOrange in non-stretched samples. Results In healthy airways PZ inhibition decreased iron uptake while activation increased intracellular iron uptake. In healthy airways, mechanical stretch upregulated SLC7A11 expression in a PZ1 independent manner, while mechanical stretch in asthmatic patients displayed no increase in the cysteine transporter. Transferrin levels remained unchanged under mechanical stimulation but displayed upregulation in asthmatic cells. In healthy cells, ferritin showed no changes in response to stretch, however, asthmatic cells showed lower baseline expression and increased its expression in response to stretch in a PZ1 independent manner. GPX4 showed no changes in expression between healthy and asthmatic groups or in response to stretch. Conclusions Our findings suggest that PZ and mechanical stretch play a role in iron regulation in hASMs. Additionally, iron regulation is impaired in asthmatic populations, suggesting a resultant increased iron import, decreased capacity to sequester iron, and decreased defensive cysteine import. This abstract is funded by: NIH R01HL171915 (CMP), R01HL056470 (YSP) 24POST1241979 (NAB)