Hamrick, Samantha K.

siRNA delivery to target cellular processes is an appealing area in lung diseases such as asthma. To realize this potential requires efficient, targeted cytosolic delivery without cellular toxicity or siRNA degradation. We demonstrate the efficacy of PONI-Guan nanoparticles in accessing airway epithelial and smooth muscle cells with specific siRNA targeting of mRNAs of interest while maintaining barrier integrity and avoiding cellular toxicity.

Iron is an essential element for cellular homeostasis and functionality. Increased iron in the lung is associated with aging and asthma in the elderly (AIE). Using human airway smooth muscle cells from young versus elderly versus AIE patients, we found that dysregulated iron metabolism (more than iron accumulation), resulting in activation of antioxidant systems and increased susceptibility to ferroptosis, occurs in AIE, contributing to cell hyperplasia and matrix remodeling of aging and AIE.

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 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 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)

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.

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.