Thompson, Martin W.

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 involves airway inflammation, hyperresponsiveness (AHR), and remodeling (cell proliferation and fibrosis), with both aspects not fully alleviated by current therapies. GPCR-based drugs represent a major aspect of current asthma therapy. Asthmatic airways express several orphan receptors with unknown physiological roles, highlighting a potentially novel and significant area for therapeutic exploration. Herein, we focus on orphan retinoic acid (RA)-induced Class C GPCR members GPRC5A (RAIG1; 5A) and GPRC5B (RAIG2; 5B). We leveraged recently identified 5A agonists to test the hypothesis that these GPCRs have alleviating effects in airways. Methods: Primary human airway smooth muscle cells (ASM) originating from non-asthmatic (NA) and asthmatic (Asth) individuals were obtained post-surgery from lung tissues after approval by the Mayo Clinic Institutional Review Board. PRESTO-Tango assay was used to determine 5A and 5B activity. cADDis assay was used to assess cAMP levels. Fluorescence imaging was used to determine calcium regulation. Recently identified 5A agonists [tryptamine, 7 fluoro-tryptamine (7FT), L-tryptophan, N-methyltryptamine, and indole-3-acetic acid] were used in assays, along with RA and 5A vs. 5B siRNAs. Results: PRESTO-Tango assay showed that tryptamine (10/100 µM) and 7FT (1/10/100 µM) increased 5A activity while high concentrations of 7FT (100 µM) increased 5B activity as well. cADDis assay showed 100 µM tryptamine increased cAMP more than 1/10 µM tryptamine and that 10 µM 7FT increased cAMP more than 1/100 µM 7FT in NA ASM.Calcium imaging showed that 5A agonist blunts responses to histamine, especially in Asth ASM. Calcium responses were higher in Asth ASM and 5A siRNA-treated ASM while 5B siRNA had an alleviating effect. Conclusions: Class C GPCRs 5A and 5B interestingly have opposing effects in human ASM towards blunting calcium responses to bronchoconstrictor agonists in the context of asthma. Thus, 5A agonists and 5B antagonists may represent novel GPCR-based approaches in airway diseases.

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: 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: 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.