Pfeffer-Kleemann, D

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 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 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 Rationale: In premature infants, continuous positive airway pressure (CPAP) serves as an effective respiratory support strategy in the NICU. However, the static stretch induced by CPAP may negatively impact bronchial airway development, leading to hyperreactivity and remodeling, key proponents of asthma. Here, epithelial and airway smooth muscle cells are affected by stretch, but the mechanisms by which effects occur are not well-known. In airway smooth muscle contractility, stromal interaction molecule 1 (STIM1) at the endoplasmic reticulum and plasma membrane Orai1 channels are important mediators of the store operated calcium entry (SOCE) microdomain signaling pathway, facilitating movement of calcium into the cell and triggering downstream remodeling effects. Recently discovered mechanosensitive Piezo (PZ) channels play a critical role in calcium influx upon mechanical stimulation. In this study, we investigated the hypothesis that interplay between mechanosensitive channels such as PZ and the SOCE microdomain promotes calcium in human fetal airway smooth muscle cells (fASM) towards increasing contractility by stretch. Methods: fASM were obtained from 18-22 week abortus following fetal demise (exempt per Mayo IRB). Cells were pretreated with either PZ1 inhibitor GsMTx4 [1uM] or PZ2 inhibitor D-GsMTx4 [1uM] 45 minutes before stretch. Cells were exposed to normal breathing stretch conditions on a FlexCell System at a 5% cyclic strain (40 cycles/minute) and CPAP with static stretch superimposed for 24 hours. Protein expression of PZ channels and key SOCE proteins were quantified. SOCE responses of stretched and control cells were determined using established protocols in fura-2 loaded cells using fluorescence microscopy. Results: Mechanical stretching of fASM increased protein expression of PZ1, STIM1, and Orai1 in a dose dependent manner. Stretch increased intracellular SOCE responses compared to non-stretched controls. PZ1 antagonist GsMTx4 and PZ2 antagonist D-GsMTx4 inhibited stretch-induced Piezo1, STIM1, and Orai1 upregulation. GsMTx4 and D-GsMTx4 blunted stretch and non-stretched induced calcium SOCE responses relative to unstretched control. Conclusions: Our findings suggest that in the context of mechanical stretch, Piezo channels and the downstream SOCE signaling pathway increase in expression and functionality in the fetal airway which may contribute to CPAP associated changes in airway contractility.

Abstract Rationale: With an increasing elderly population, understanding mechanisms that contribute to structural and functional changes in aging lung is important in the context of healthy aging and diseases such as asthma and pulmonary fibrosis that disproportionately affect the elderly. Asthma in the elderly (AIE) is a particular challenge, with thickened fibrotic bronchial airways (remodeling) and inflammation compared to non-asthmatic elderly: issues not addressed by current therapies. Factors such as iron regulation that are critical for normal physiology can become dysregulated in disease. Iron overload can catalyze a Fenton reaction that increases ROS which oxidizes lipids and proteins, i.e. iron-mediated lipid peroxidation cell death (ferroptosis).The goal of this study was to determine iron regulation in aging- and AIE-associated in human ASM (hASM), a cell type important in airway contractility and remodeling. Methods: hASM cells and lung tissues from young (<45y), and aged (>65y) male/female patients with/without asthma were used (approved by Mayo IRB #16-009655). Cells were treated with ferroptosis inhibitor deferoxamine (DFO) [100µM], inducer RSL3 [125 nM] or vehicle (DMSO) for 24 hrs. Levels of intracellular iron (Fe2+) were determined using FerroOrange. Cell lysates were examined for iron accumulation and ferroptosis markers (4-HNE, FTH1, TfR1, SLC7A11, and KEAP1), cell proliferation, and ECM production (COL1A1 and FN1), and supernatants analyzed for proinflammatory mediators IL-6 and IL-8. Results: Non-asthmatic and asthmatic elderly hASM cells showed a higher cytosolic Fe2+ and lower end-product of lipid peroxidation (4-HNE), higher level of SLC7A11 and KEAP1 (regulators of redox systems) compared to cells from young individuals. High level of iron storage (FTH1) was observed in asthmatics compared to non-asthmatic elderly, but no difference was observed for iron transporters (TfR1). Furthermore, COL1A1 and FN1 were higher in AIE hASM, compared to young hASM. COL1A1 was higher in aging hASM compared to young. DFO reduced COL1A1 in elderly, but upregulated in AIE. Cell proliferation was inhibited by DFO in aging hASM but not in young and AIE. The ferroptosis inducer RLS3 impaired antioxidant system and increased lipid peroxidation and IL-8 levels. Conclusions: Our findings suggest that iron regulation is impaired in aging and AIE hASM, resulting in iron accumulation which modulates airway remodeling via cell proliferation and ECM production. While ferroptosis per se can be targeted, it will be important to explore modulation of specific iron regulatory pathways in alleviating the impact of aging and asthma.