Prakash, Y. S.

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.

Abstract Rationale : Airway remodeling in asthma involves increased airway smooth muscle (ASM) mass, changes to bronchial epithelial cell (BEC) layer, and increased fibrosis. ASM and BECs actively contribute by secreting growth factors and cytokines, regulating extracellular matrix (ECM) composition and enhancing cell proliferation/migration. Altering mechanisms which modulate remodeling presents a key to novel therapeutic strategies. BDNF increases ASM [Ca2+]i and contractility, and potentiates the effects of inflammation. Importantly, studies show that ASM is not only a target, but also a source of BDNF, raising the possibility of local autocrine/paracrine effects. Arginase 1 and 2 are expressed by BECs, but their roles in asthma are still under investigation with studies showing arginase 1 inhibition blunting epithelial remodeling and ASM hyperplasia. Knockdown of appealing targets such as ASM BDNF and epithelial arginases using small interfering RNA (siRNA) is a promising approach to combating inflammatory lung remodeling. Polymer based nanoparticles have been constructed which demonstrate cell-type specificity and high efficiency in delivering siRNA. Thus, we hypothesize that BDNF and arginases are central to airway remodeling and can be targeted via nanopartiacles.Methods: Primary human ASM and BEC cells from non-asthmatic vs. asthmatic individuals were obtained post-surgery from lung tissues after approval Mayo Clinic IRB. PONI-Guan NP were generated using block copolymer engineering. PCR, immunoblotting and ELISA were performed using standard techniques. Contractility studies were conducted in adult mouse precision cut lung slices. In vivo lung function studies were conducted in 6-8 week old mice, with or without mixed allergen (MA) treatment and Scireq Flexivent system. Results: PONI-Guan NP uptake demonstrates cell-type specificity in BEC/ASM co-culture and mouse lung. Arginase I/II and ECM expression is upregulated in BECs when exposed to BDNF and cytokines. In ASM, BDNF is increased in asthmatics compared to non-asthmatic controls; ECM expression is increased in response to BDNF and TNF-α. In BEC, PONI-Guan NP/siRNA inhibits Arg I/II expression, even in the presence of TNF-α. In ASM, TNF-α increases in BDNF secretion and ECM deposition are blunted by NP-delivered siRNA. PCLS and mouse lung function studies show contractility to MCh: effects suppressed by NP-delivered BDNF and Arg I siRNA. Conclusions: Our studies show the importance of BDNF/Arginase pathways in airway remodeling and efficacy of PONI-based siRNA delivery vectors in blunting remodeling processes.

Abstract RATIONALE: Endothelial-to-mesenchymal transition (EndMT) is a cellular process in which endothelial cells (ECs) transition into mesenchymal cells, a mechanism recently linked to pulmonary vascular remodeling in pulmonary hypertension (PH). However, the regulatory mechanisms driving EndMT in PH remain largely undefined. MicroRNA-153 (miR-153), a small non-coding RNA, regulates gene expression by targeting the 3'-untranslated region (3'-UTR) of mRNA, affecting mRNA translation and stability. This study investigates miR-153's role in modulating EndMT in PH. METHODS: Human lung vascular endothelial cells (LVECs) were isolated from healthy individuals and patients with idiopathic pulmonary arterial hypertension (IPAH). In vitro, normal LVECs were exposed to hypoxia (3% O₂) for 72 hours or treated with TGF-β1 (10 ng/mL) for 7 days to induce EndMT. Levels of miR-153 and EndMT markers were quantified by qPCR, Western blotting, or immunostaining. Proliferation and apoptosis of ECs were assessed using EdU and TUNEL assays, respectively. LVECs were transfected with miR-153 inhibitor or mimic, then exposed to normoxic or hypoxic conditions, respectively for 72 hours. In vivo, mice were subjected to either hypoxia (10% O₂ for 6 weeks, Hx-PH) or Sugen5416/hypoxia (20 mg/kg i.p. plus 10% O₂ for 6 weeks, SuHx-PH) to induce PH. The PH phenotype in mice was evaluated by hemodynamic measurements and histological analysis of H&E-stained lung tissues. RESULTS:In silico analysis identified miR-153 as a direct regulator of human SNAI1 and SNAI2 genes via binding to their 3'-UTR. Downregulation of miR-153 in IPAH LVECs was correlated with elevated SNAI1/2 expression, increased EndMT, and higher proliferation compared to normal LVECs. Hypoxia or TGF-β1 treatment reduced miR-153 expression, increased EndMT transcription factors (SNAI1/2), mesenchymal markers (SM-22, vimentin), and reduced EC markers (CD31, VE-cadherin). In normoxic LVECs, miR-153 inhibition significantly upregulated SNAI1/2, increased EndMT and EC proliferation, and reduced apoptosis. Conversely, miR-153 mimic abolished hypoxia- and TGF-β1-induced EndMT. Overexpression of miR-153 attenuated hypoxia-induced EC survival. In PH mouse models, reduced miR-153 levels were associated with activated SNAI1/2, EndMT, and pulmonary vascular remodeling. CONCLUSIONS: Downregulation of miR-153 drives phenotypic transition of LVECs to proliferative mesenchymal cells via SNAI-mediated EndMT, contributing to pulmonary vascular remodeling and PH. Therefore, miR-153 could be a potential therapeutic target for PH treatment.

EndMT has been reported to contribute to the pathogenesis of PAH. In this study, we aimed to determine the role of Ca 2+ signaling in the development of EndMT in human lung vascular endothelial cells. Our data suggest that TGF-β 1 requires store-operated Ca 2+ entry through STIM1/Orai channels to induce SNAI-mediated EndMT. For the first time, we demonstrated that TGF-β 1 -induced EndMT is a Ca 2+ -dependent event, whereas inhibition of STIM1/Orai interaction attenuated EndMT in response to TGF-β 1 .

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 The lung is an inherently mechanosensitive organ, where cells of the airway and parenchyma experience a range of mechanical forces throughout life including shear, stretch, and compression, in both health and disease. In this regard, pediatric and adult lung diseases such as wheezing and asthma, bronchopulmonary dysplasia (BPD), chronic obstructive pulmonary disease (COPD), and pulmonary fibrosis (PF) all involve macroscopic and cellular changes to the mechanical properties of the bronchial airways and/or parenchyma to varying extents. Accordingly, understanding how mechanical forces are sensed in the lung, and the responses of cells and tissues in the context of normal development and health versus disease conditions becomes highly relevant. There is increasing recognition that transduction of mechanical forces into cellular responses involves a number of channels, some of which are inherently mechanosensitive. Such channels trigger mechanotransduction pathways that may further mediate cellular remodeling, inflammation, and other pathophysiologic mechanisms in response to stretch, stiffness, and inflammatory cascades. Two particularly important channel families have emerged in pulmonary pathophysiology: the transient receptor potential vanilloid family with focus on member TRPV4 and the recently identified Piezo (PZ) channels. Here, we explore current understanding of the contributions of TRPV4 and PZ channels in lung health and disease states, focusing on the interactions between these mechanosensitive channels and their local environment including immune cells, the extracellular matrix, and cellular cytoskeletal elements. We further discuss potential areas for future research to better understand the impact of mechanical channels on pulmonary health and disease. © 2023 American Physiological Society. Compr Physiol 13:5157‐5178, 2023.

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.

Asthma is a heterogeneous chronic lung disease that affects nearly 340 million people globally. Airway hyperresponsiveness, remodeling (thickening and fibrosis), and mucus hypersecretion are some hallmarks of asthma. With several current treatments having serious side effects from long-term use and a proportion of patients with uncontrolled asthma, there is an urgent need for new therapies. With an increasing understanding of asthma pathophysiology, there is a recognized need to target therapies to specific cell types of the airway, which necessitates the identification of delivery systems that can overcome increased mucus and thickened airways. Nanoparticles (NPs) that are highly customizable (material, size, charge, and surface modification) are a potential solution for delivery systems of a wide variety of cargoes (nucleic acids, proteins, and/or small molecules), as well as sole therapeutics for asthma. However, there is a need to consider the safety of the NPs in terms of potential for inflammation, toxicity, nonspecific targets, and accumulation in organs. Ongoing clinical trials using NPs, some FDA-approved for therapeutics in other diseases, provide confidence regarding the potential safety and efficacy of NPs in asthma treatment. This review highlights the current state of the use of NPs in asthma, identifying opportunities for further improvements in NP design and utilization for targeting this chronic lung disease.