Ngassie, Maunick Lefin Koloko

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.

Mechanical stretch associated with respiratory support can impair airway development and function in neonates, but the mechanisms are not fully understood. Using developing human airway smooth muscle cells exposed to cyclic forces with static stretch to mimic continuous positive airway pressure, we found that stretch dysregulates long-term cell proliferation and inflammatory cytokine production, and mechanosensitive Piezo ion channels may play a role in the proliferation response.

Abstract Rationale: While survival of premature infants has improved with NICU interventions, prematurity remains a risk for developing chronic lung diseases. Oxygen supplementation in the NICU is unfortunately necessary, and clinical practice has shifted towards use of moderate hyperoxia (<60% O2) to minimize detrimental effects of very high O2 concentration (80-90%) such as bronchopulmonary dysplasia. However, even moderate hyperoxia increases the risk of bronchial airway diseases and wheezing/asthma with airway hyperreactivity and remodeling. We previously found that moderate hyperoxia induces cellular senescence in human fetal airway smooth muscle cells (fASM) and fibroblasts. However, the mechanisms by which hyperoxia enhances cellular senescence in fASM are not known. We investigated the role of endoplasmic reticulum (ER) stress and mitochondrial dysfunction, using fASM cells exposed to 21% (normoxia) vs. 50% O2 (hyperoxia). Methods: fASM isolated from lungs of 18-20 week aborted fetuses (Mayo IRB exempt; period of rapid bronchial airway growth) were exposed to normoxia, hyperoxia or etoposide (10 µM; positive control for senescence induction). Cells and lysates were harvested at days 2, 3 or 7 to assess markers of senescence markers, ER stress and mitochondrial morphology, and levels of cytosolic and mitochondrial ROS, using real-time fluorescence imaging, Western blotting, immunofluorescence, and a novel immuno-FISH staining of telomere associated foci (TAF). Mitochondria-specific antioxidant MitoQ [100 nM], ER stress inhibitor Salubrinal [12.5 µM] or mitochondrial fission inhibitor Mdivi-1 [10 µM] were used to assess their effects on senescence markers and interactions between ER stress and mitochondria in inducing senescence. Results: Hyperoxia enhanced cellular senescence via upregulation of p21, PAI-1, and Cav-1; downregulation of LMNB1 and Ki-67, with increased γH2A.X- and TAF-positive cells. Hyperoxia increased cytosolic and mitochondrial ROS, and markers of the three ER stress pathways (ATF6, IRE1α, XBP1s, PDI, GADD34). Hyperoxia altered mitochondrial morphology with increased fission protein Drp1 and fusion proteins Mfn1/2. Inhibition of Drp1 with Mdivi-1 blocked mitochondrial fragmentation, but not senescence. However, hyperoxia-induced p21 was reduced by the PERK pathway inhibitor Salubrinal and MitoQ. Conclusions: Hyperoxia induces senescence in fASM via mitochondrial ROS and ER stress: pathways that offer new opportunities to blunt detrimental effects of oxygen in developing lung via mitochondrial targeted antioxidants and/or ER stress inhibition.

Abstract Rationale: Asthma in the elderly (AIE) presents a unique challenge characterized by hyperreactive and thickened, fibrotic airways (remodeling) that is not adequately addressed by current therapies. Thus, understanding mechanisms that drive AIE become 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) in health and disease. The lung generates mechanical forces of normal breathing and other maneuvers that are transmitted to bronchial airways via tethering parenchyma. While Pz channels are highly expressed in the lung, their specific role in ASM function remains largely unexplored. In this study, we used human ASM cells to test the hypothesis that increased airway stiffness associated with normal aging blunts Pz expression and mechanosensitive functionality, whereas asthmatic triggers (e.g. inflammation) enhance Pz expression and downstream signaling. Methods: Primary human ASM cells isolated from lungs of young (<45y) vs. aged (>65y) non-asthmatic vs. asthmatic individuals were obtained post-surgery (Mayo Clinic IRB approved). Cells were exposed to mechanical stretch (mimicking breathing) and mediators relevant to asthma (TNFα, IL-13) or fibrosis (TGFβ) to determine expression of Pz1/2 across the age spectrum. Separately, intracellular Ca2+ and contractility responses, proliferation and extracellular matrix (ECM) deposition across age/asthma groups were assessed in response to mechanical stretch and Pz1/2 modulation (inhibitors/activators, genetic approaches). Manipulation of Pz1/2 expression or function in ASM accompanied by mechanical stretch was used to assess impact on canonical Wnt/β-catenin signaling. Results: We observed increased Pz1/2 expression with age and asthma as well as with inflammation and mitogenic stimuli. Functionally, Pz1 agonist, Yoda1 induced increase in intracellular Ca2+, ASM proliferation and ECM deposition with greater effects observed in aging asthmatic ASM. Towards linking mechanosensation (via Pz) to remodeling via Wnt signaling, we find that Wnt3a is upregulated by Yoda1 but suppressed by Pz1 shRNA. Yoda1 stimulates TGFβ-induced Wnt3a and β-catenin, an effect further enhanced under mechanical stretch. Conclusions: By elucidating the role of Pz channels in ASM, we aimed to identify novel therapeutic targets for airway diseases. Understanding how mechanical forces and inflammatory stimuli influence Pz-mediated signaling pathways will lead to the development of innovative strategies to prevent or reverse airway remodeling in elderly asthmatic patients. Acknowledgement: Supported by 24POST1241979 (NAB) and NIH grants R01-HL142061 (CMP and YSP), R01- HL088029 (YSP).

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.

Supplemental O 2 (hyperoxia) in premature infants detrimentally affects bronchial airways leading to increased senescence. Understanding the mechanisms by which hyperoxia initiates senescence in developing airways is critical for future therapeutic strategies. The current study showed that hyperoxia-induced senescence is mediated through increased mitochondrial reactive oxygen species and endoplasmic reticulum (ER) stress. ER stress inhibitors or mitochondria-targeted antioxidants may represent future therapies to blunt detrimental effects of supplemental oxygen in developing lungs.

Abstract Human chromosomal anomalies, notably trisomies, disrupt gene expression, leading to diverse cellular and organ phenotypes. Increased cellular senescence (SEN) and oxidative stress in trisomies have gained recent attention. We assessed SEN, SEN-associated secretory phenotype (SASP), and oxidative stress on trisomy 13 (T13), T18, and T21 human fetal lung tissues and isolated primary human fetal lung fibroblasts. Telomerase-associated foci staining showed DNA damage primarily within T21 and T18 lungs. These results were confirmed by real-time quantitative PCR showing an increase of the SEN marker CDKN2B and SASP markers IL-6 and CXCL8. In contrast, lung tissues from T13 showed an upregulation of CDKN2A, whereas no significant changes in SASP marker genes were observed. γ-H2AX (H2A histone family member X) was upregulated in each genotype, particularly in T21. Isolated fibroblasts demonstrated a strong association between T21 and several SEN markers. An increase of γ-H2AX–positive cells were observed in fibroblasts from T21, T18, and T13, but only T21 exhibited an increase in P21 expression. Only T21 fibroblasts displayed a significant increase in reactive oxygen species levels, as indicated by MitoSOX and CellROX. This study provides the first evidence of a link between SEN and trisomy anomalies during prenatal human lung development, particularly in T21.

Our previous study suggests that glial-derived neurotrophic factor (GDNF) is involved in allergen-induced airway hyperreactivity and remodeling in vivo. Here, we show that GDNF has no direct effects in remodeling of human airway smooth muscle (ASM) but GDNF dysregulates mitochondrial morphology in human ASM in the context of asthma.

Our study identified chronic obstructive pulmonary disease (COPD)-associated differences in the lung extracellular matrix (ECM) composition. We highlight the compartmental differences in the ECM landscape in different subtypes of COPD. The most prominent differences were observed for severe-early onset COPD. Moreover, we identified unique ECM signatures that describe airway walls and parenchyma providing insight into the intertwined nature and complexity of ECM changes in COPD that together drive ECM remodeling and may contribute to disease pathogenesis.

Mechanical forces on the airway can contribute to altered contractility and remodeling in airway diseases, but the mechanisms are not clearly understood. Using human airway smooth muscle cells exposed to cyclic forces with static stretch to mimic breathing and static pressure, we found that the effects of stretch are mediated through STIM1, resulting in the activation of multiple pathways, including Piezo channels and the inflammasome, with potential downstream influences on contractility and remodeling.