Prakash, Y. S.

The pathogenesis of asthma in elderly is not well understood. Using a mouse model of asthma, we show that aging results in decreased lung function and less responsiveness to allergen exposure, impacted by locally produced brain-derived neurotrophic factor. Aging also decreases allergen-induced inflammation but increases airway remodeling and senescence. Our results suggest that senescence pathways may contribute to asthma pathogenesis in elderly.

Key Points PIEZO1 activation reduces cystogenesis: Yoda1 activates PIEZO1, raising calcium and lowering cAMP, reducing cyst growth in autosomal dominant polycystic kidney disease models. Context-dependent role of PIEZO1: PIEZO1 knockout in mice with or without polycystic kidneys does not affect cyst formation, suggesting redundancy in mechanosensitive pathways. Therapeutic potential: Findings support PIEZO1 activation as a part of combination therapy to slow cyst growth in autosomal dominant polycystic kidney disease, needing more in vivo studies. Background The disruption of calcium signaling associated with polycystin deficiency is a key factor in abnormal epithelial growth in autosomal dominant polycystic kidney disease. Calcium homeostasis can be influenced by mechanotransduction. The mechanosensitive cation channel PIEZO1 has been implicated in sensing intrarenal pressure and regulating urinary osmoregulation, but its role in kidney cystogenesis is unclear. Methods We hypothesized that altered mechanotransduction contributes to cystogenesis in autosomal dominant polycystic kidney disease and that activation of mechanosensitive cation channels could be a therapeutic strategy. Results We demonstrate that Yoda1, a PIEZO1 activator, increases intracellular calcium and reduces forskolin-induced cAMP levels in mouse inner medullary collecting duct (mIMCD3) cells. Notably, knockout of polycystin-2 attenuated the efficacy of Yoda1 in reducing cAMP levels in mIMCD3 cells. Yoda1 also reduced forskolin-induced mIMCD3 cyst surface area in vitro and cystic index in mouse metanephros ex vivo in a dose-dependent manner. However, collecting duct–specific PIEZO1 knockout neither induced cystogenesis in wild-type mice nor altered cystogenesis in the Pkd1 RC/RC mouse model. Conclusions These findings support the potential role of PIEZO1 agonists in mitigating cystogenesis by increasing intracellular calcium and reducing cAMP levels, but the unaltered in vivo cystic phenotype after PIEZO1 knockout in the collecting duct suggests possible redundancy in mechanotransductive pathways.

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.

Abstract Nicotine exposure in the context of smoking or vaping worsens airway function. Although nicotinic acetylcholine receptors (nAChRs) are commonly thought to exert effects through the peripheral nervous system, we previously showed that airway smooth muscle (ASM) expresses them, particularly α7 subtype nAChR (α7nAChR), with functional effects on contractility and metabolism. However, the mechanisms of nAChR regulation and downstream effects in ASM are not fully understood. Using ASM cells from people without asthma versus people with mild to moderate asthma, we tested the hypothesis that nAChR-specific endoplasmic reticulum (ER) chaperones, resistance to inhibitors of cholinesterase 3 (RIC-3) and transmembrane protein 35A (TMEM35A) promote cell surface localization of α7nAChR with downstream influence on its functionality: effects exacerbated by inflammation. We found that mild to moderate asthma and exposure to proinflammatory cytokines relevant to asthma promote chaperone and α7nAChR expression in ASM. Downstream, ER stress was linked to nicotine/α7nAChR signaling, where RIC-3 and TMEM35 regulate nicotine-induced ER stress, intracellular Ca2+ regulation, and ASM cell proliferation. Overall, our data highlight the importance α7nAChR chaperones in mediating and modulating nicotine effects in ASM toward airway contractility and remodeling.

ABSTRACTInflammatory cell infiltration is a characteristic feature of COPD and correlates directly with the severity of the disease. Interleukin‐23 (IL‐23) is a pro‐inflammatory cytokine that regulates Th‐17 inflammation, which mediates many pathophysiological events in COPD. The primary goal of this study was to determine the role of IL‐23 as a mediator of key pathologic processes in cigarette smoke‐induced COPD. In this study, we report an increase in IL23 gene expression in the lung biopsies of COPD patients compared to controls and identified a positive correlation between IL23 gene expression and disease severity. In a cigarette smoke‐induced murine emphysema model, the suppression of IL‐23 with a monoclonal blocking antibody reduced the severity of cigarette smoke‐induced murine emphysema. Mechanistically, the suppression of IL‐23 was associated with a reduction in immune cell infiltration, oxidative stress injury, and apoptosis, suggesting a role for IL‐23 as an essential immune mediator of the inflammatory processes in the pathogenesis of CS‐induced emphysema.

The lung is an inherently mechanosensitive organ that can respond to mechanical forces in adaptive or maladaptive ways, including via remodeling resulting in increased fibrosis. We explored the mechanisms that link mechanical forces to remodeling using human lung fibroblasts. We found that mechanosensitive Piezo channels increase with stretch and mediate extracellular matrix formation and the fibroblast-to-myofibroblast transition that occurs with stretch. Our data highlight the importance of Piezo channels in lung mechanotransduction toward remodeling.

Fibrosis in the lung is thought to be driven by epithelial cell dysfunction and aberrant cell-cell interactions. Unveiling the molecular mechanisms of cellular plasticity and cell-cell interactions is imperative to elucidating lung regenerative capacity and aberrant repair in pulmonary fibrosis. By mining publicly available RNA-Seq data sets, we identified loss of CCAAT enhancer-binding protein alpha (CEBPA) as a candidate contributor to idiopathic pulmonary fibrosis (IPF). We used conditional KO mice, scRNA-Seq, lung organoids, small-molecule inhibition, and potentially novel gene manipulation methods to investigate the role of CEBPA in lung fibrosis and repair. Long-term (6 months or more) of Cebpa loss in AT2 cells caused spontaneous fibrosis and increased susceptibility to bleomycin-induced fibrosis. Cebpa knockout (KO) in these mice significantly decreased AT2 cell numbers in the lung and reduced expression of surfactant homeostasis genes, while increasing inflammatory cell recruitment as well as upregulating S100a8/a9 in AT2 cells. In vivo treatment with an S100A8/A9 inhibitor alleviated experimental lung fibrosis. Restoring CEBPA expression in lung organoids ex vivo and during experimental lung fibrosis in vivo rescued CEBPA deficiency–mediated phenotypes. Our study establishes a direct mechanistic link between CEBPA repression, impaired AT2 cell identity, disrupted tissue homeostasis, and lung fibrosis.

AbstractSupplemental O2 (hyperoxia) is a critical intervention for premature infants (<34 weeks) but consequently is associated with development of bronchial airway hyperreactivity (AHR) and asthma. Clinical practice shifted toward the use of moderate hyperoxia (<60% O2), but risk for subsequent airway disease remains. In mouse models of moderate hyperoxia, neonatal mice have increased AHR with effects on airway smooth muscle (ASM), a cell type involved in airway tone, bronchodilation, and remodeling. Understanding mechanisms by which moderate O2 during the perinatal period initiates sustained airway changes is critical to drive therapeutic advancements toward treating airway diseases. We propose that cellular clock factor BMAL1 is functionally important in developing mouse airways. In adult mice, cellular clocks target pathways highly relevant to asthma pathophysiology and Bmal1 deletion increases inflammatory response, worsens lung function, and impacts survival outcomes. Our understanding of BMAL1 in the developing lung is limited, but our previous findings show functional relevance of clocks in human fetal ASM exposed to O2. Here, we characterize Bmal1 in our established mouse neonatal hyperoxia model. Our data show that Bmal1 KO deleteriously impacts the developing lung in the context of O2 and these data highlight the importance of neonatal sex in understanding airway disease.

Rationale: Lung associated diseases are one of the leading causes of death in Down syndrome/Trisomy21 (DS/T21). DS is often considered a progeroid syndrome, associated with increased cellular senescence and oxidative stress in dermal T21 fibroblasts. Senescence plays a crucial role in development and organogenesis. We recently demonstrated that T21 lung anomalies are initiated in utero. We aim to better understand the role of senescence in lung disease in T21. Methods: Fresh T21 and, age- and sex-matched non-T21 human fetal lungs were collected, dissociated, and human fetal lung fibroblast (HFLF) cultures were established using differential adhesion. Baseline senescence levels were determined in whole tissue and isolated fibroblasts using TAF staining for tissue only and RT-qPCR and immunofluorescence (IF) for both. β-galactosidase, CellROX/MitoSOX, and calcium release (FURA2/AM imaging) assays were performed on T21 and non-T21 fibroblasts, cultured in the absence or presence of an ER stress inhibitor (4-PBA, 2mM) or a mitochondriogenesis stimulator (PQQ, 20μM). Results: Whole tissue sections demonstrated increased TAF and γ-H2AFX staining in T21 vs non-T21. RT-qPCR demonstrated significantly increased expression of several senescence markers including CDKN1A, CDKN2A, TP53, CDKN2B and PAI1 (p<0.05; n=6) at baseline in T21 HFLF as compared to non-T21. IF for γ-H2AFX and P21 were also significantly increased in T21 cultures vs non-T21 (p<0.01, n=6). Upregulated senescence was further confirmed in T21 HFLF by increased β-galactosidase activity (6-fold vs non-T21, p<0.05, n=4) as well as increased ER and mitochondria stress markers (CellROX and mitoSOX, p<0.05, n=6) in T21 cultures. Fura2/AM assay revealed that T21 HFLF exhibit greater intracellular calcium release (p<0.01, n=3) than non-T21 HFLF at both baseline and post Acetylcholine (10uM) challenge, an indicator of increased ER stress. In addition, several mitochondria morphology genes ( FIS1, DNML1 and MFN1) increased in T21 HFLF (p<0.05, n=6). Both 4-PBA or PQQ treatments reversed senescence in T21 HFLF as shown by decreased β-galactosidase staining (p<0.01; n = 4). No differences were observed in non-T21 HFLF after treatment. However, gene expression analyses of CDKN1A, CDKN2A, TP53 and CDKN2B demonstrated attenuated expression in T21 HFLF only following PQQ treatment but not 4-PBA (p<0.05, n=4). Conclusion: In this study, we established that fibroblasts from T21 lungs present with both ER and mitochondria-associated senescence. However, only PQQ, the mitochondriogenesis stimulator, was able to completely reverse the senescence observed at baseline in T21 HFLF. Understanding this increase in senescence in the lungs of individuals with Down Syndrome may help address the respiratory issues that arise after birth. CIRM (EDUC4-12837) Postdoctoral Training Grant (RB) NIH/NHLBI R01HL141856 (DA); NIH/NHLBI Offce of The Director, National Institutes of Health (OD) R01HL155104 (SD); NIH/NHLBI R21HL165411 (SD and DA). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

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.