Citations

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)

Rationale: Pulmonary hypertension (PH) is a fatal disease characterized by a sustained elevated pulmonary vascular resistance that results in right heart failure. Despite therapeutic advances, no treatment provides disease progression reversion, resulting in poor long-term survival. Identifying new therapeutic targets for PH therefore requires a deeper understanding of its mechanisms. A defining feature of PH is pulmonary vascular remodeling, a result from excessive proliferation, migration, and apoptosis resistance of pulmonary artery smooth muscle cells (PASMC). SNAI1, a transcription factor regulating cell proliferation and differentiation during development and disease, has recently been implicated in endothelial dysfunction and endothelial-to-mesenchymal transition in PH. However, its role within PASMC remains unknown. Here, we aimed to define the contribution of SNAI1 in PASMC to the pathogenesis of PH. Methods: Normal human PASMC were exposed to normoxia (21% O 2 ) or hypoxia (3% O 2 ) for 72 hours. PASMC were also isolated from patients with or without PH. Cell proliferation and migration were assessed by EdU incorporation and wound scratch assays respectively. Mice were exposed to hypoxia (10% O 2 ) or Sugen5416/hypoxia (10% O 2 plus Sugen5416 20mg/kg, i.p., weekly) for 6 weeks. PH phenotype was characterized by right heart catheterization, right ventricle (RV) hypertrophy by Fulton index and pulmonary artery (PA) remodeling by H&E staining of lung tissues. Pharmacological blocker GN-25 was used to inhibit SNAI1 in vitro and in vivo. Results: SNAI1 expression was markedly increased in hypoxic PASMC and in PASMC from patients with PH. Hypoxia also upregulated HIF-1alpha, Vimentin, p-AKT, PCNA at the protein level in PASMC. EdU incorporation and migration rates were significantly higher in hypoxic cells as well as in PASMC isolated from PH patients compared with controls. In vitro, GN-25 (100 nM) abolished hypoxia-induced PASMC proliferation and migration. Right ventricular systolic pressure (RVSP), a surrogate measurement of PA systolic pressure, estimated mean PAP (mPAP), and RV contractility (RV-±dP/dtmax) were significantly increased in both PH models compared with controls. Additionally, Sugen5416/hypoxia-treated mice exhibited higher RVSP, mPAP and RV-±dP/dtmax compared to hypoxia-treated animals. The Fulton index (weight of RV vs. left ventricle and septum), was increased in both PH groups indicating development of RV hypertrophy in PH compared to control. PH mice lost body weight by 5-10%. Additionally, when Fulton index was normalized by body weight, RV hypertrophy was significantly higher in Sugen5416/hypoxia-treated mice compared with hypoxic animals. Lung tissues from PH mice exhibit PA hypertrophy (media remodeling) with some PA partially or completely occluded. Additionally, we detected protein upregulation of Snai1 and Vimentin in lung tissues of PH mice in both groups. Treatment of PH mice with GN-25 (20 mg/kg, daily, for the last 3 weeks under PH stimuli) in reversal experiments attenuated PH phenotype in both animal models by decreasing hemodynamic parameters, right ventricle hypertrophy and pulmonary vascular remodeling. Conclusions: Our findings identify a key role for SMC SNAI1 in the pathogenesis of lung vascular remodeling during hypoxia-induced PH. Inhibition of SNAI1 in PASMC may constitute a new therapeutic strategy for the treatment of PH. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

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 Ca 2 + 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 Ca 2 + 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 was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

ABSTRACT Supplemental oxygen (hyperoxia), often provided to premature infants, can disrupt lung growth and contribute to development of neonatal and pediatric lung diseases, necessitating understanding of underlying mechanisms. We previously showed that even moderate hyperoxia (< 60% O 2 ) induces detrimental cellular senescence in 18–22 weeks human fetal airway smooth muscle (fASM), a key cell type in airway contractility and remodeling. In this study, we examined the ability of senotherapeutics Fucoidan and Dasatinib (D) + Quercetin (Q) (D + Q) to mitigate adverse effects of hyperoxia and assessed the preventive effect of mitochondrial antioxidants (MitoQ). fASM cells exposed to normoxia (21% O 2 ) or hyperoxia (50% O 2 ) were treated with Fucoidan [100 μg/mL], D + Q [250 nM + 375 nM] or MitoQ [100 nM] and assessed for senescence markers, senescence‐associated secretory profile (SASP), extracellular matrix (ECM) deposition, and cell viability. Our results showed that moderate hyperoxia increases senescence markers and SASP. Fucoidan decreased p21 expression and inhibited SASP release without inducing cell death, while D + Q decreased β‐galactosidase (β‐Gal) activity, p21 and plasminogen activator inhibitor‐1 (PAI‐1) expression, and caused cell death without affecting SASP in hyperoxia‐induced senescent fASM. MitoQ prevented increases in senescence markers and SASP in hyperoxia‐exposed fASM. These findings demonstrate distinct and viable strategies to counter hyperoxia‐induced fASM senescence. Fucoidan (senomorphic), D + Q (senolytic), and MitoQ (prophylactic antioxidant) represent promising, mechanistically different approaches to alleviate detrimental effects of moderate hyperoxia in developing airways towards limiting perinatal lung disease.

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.

Abstract Asthma in the elderly (AIE) represents a unique challenge, necessitating better understanding of the mechanisms that drive airway hyperreactivity and remodeling (thickened, fibrotic airways). Normal aging is associated with increased lung stiffness, and thus the influence of mechanical forces on bronchial airways (breathing, sighs, CPAP) likely changes, potentially increasing with AIE and resultant remodeling, overall highlighting the need to understand the importance of mechanobiology in aging and AIE. Here, mechanosensitive Piezo (Pz) channels are highly expressed in the lung, including within airway epithelium and smooth muscle (ASM) but their specific roles particularly with aging remain largely unexplored. In this study, we used human ASM cells from young vs. old (≥65 y) non-asthmatics vs asthmatics to test the hypothesis that normal aging blunts Pz expression and functionality at baseline as well as in response to mechanical stretch, whereas asthma, particularly, AIE ASM leads to enhanced Pz expression promoting fibrosis and remodeling. Both Pz1 and Pz2 were expressed in ASM and increased in AIE. Oscillatory mechanical stretch mimicking breathing with superimposed static stretch increased Pz, especially in AIE: effects exacerbated by Pz activator Yoda1 and blunted by Pz inhibitor GsMTx4. Stretch or Yoda1 increased Wnt3a, β-catenin and GSK3β activation particularly in AIE, while ASM proliferation and extracellular matrix production remained preserved or even heightened in elderly ASM. Conversely, Pz1 inhibition by GsMTx4 potently reduced ECM deposition, particularly in AIE cells, highlighting Pz1's pivotal role in modulating ECM deposition. Overall, our findings indicate Pz1 as a mechanosensitive regulator of airway remodeling in AIE.