Prakash, Y S

We investigated the mechanisms involved in the development of airway hyperresponsiveness (AHR) following exposure of mice to halogens. Male mice (C57BL/6; 20–25 g) exposed to either bromine (Br2) or Cl2(600 or 400 ppm, respectively, for 30 min) developed AHR 24 h after exposure. Nifedipine (5 mg/kg body wt; an L-type calcium channel blocker), administered subcutaneously after Br2or Cl2exposure, produced higher AHR compared with Br2or Cl2alone. In contrast, diltiazem (5 mg/kg body wt; a nondihydropyridine L-type calcium channel blocker) decreased AHR to control (air) values. Exposure of immortalized human airway smooth muscle cells (hASMC) to Br2resulted in membrane potential depolarization ( VmAir: 62 ± 3 mV; 3 h post Br2:−45 ± 5 mV; means ± 1 SE; P < 0.001), increased intracellular [Ca2+]i, and increased expression of the calcium-sensing receptor (Ca-SR) protein. Treatment of hASMC with a siRNA against Ca-SR significantly inhibited the Br2and nifedipine-induced Vmdepolarization and [Ca2+]iincrease. Intranasal administration of an antagonist to Ca-SR in mice postexposure to Br2reversed the effects of Br2and nifedipine on AHR. Incubation of hASMC with low-molecular-weight hyaluronan (LMW-HA), generated by exposing high-molecular-weight hyaluronan (HMW-HA) to Br2, caused Vmdepolarization, [Ca2+]iincrease, and Ca-SR expression to a similar extent as exposure to Br2and Cl2. The addition of HMW-HA to cells or mice exposed to Br2, Cl2, or LMW-HA reversed these effects in vitro and improved AHR in vivo. We conclude that detrimental effects of halogen exposure on AHR are mediated via activation of the Ca-SR by LMW-HA.

Pharmacologic stimulation of a G protein–coupled receptor preferentially expressed on fibroblasts reverses experimental lung and liver fibrosis.

Altered airway smooth muscle (ASM) mass and extracellular matrix (ECM) deposition in airways are characteristic features of remodeling in asthma. Increased ECM production modulates ASM cell proliferation and leads to airway remodeling. Our previous studies showed that ASM from patients with asthma exhibited increased expression of estrogen receptor (ER)‐β, which upon activation down‐regulated ASM proliferation, implicating an important role for estrogen signaling in airway physiology. There is no current information on the effect of differential ER activation on ECM production. In this study, we evaluated the effect of ER‐α vs. ER‐β activation on ECM production, deposition, and underlying pathways. Primary human ASM cells isolated from asthmatics and nonasthmatics were treated with E 2 , an ER‐α agonist [propylpyrazoletriol (PPT)], and an ER‐β agonist [WAY‐200070 (WAY)] with TNF‐α or platelet‐derived growth factor (PDGF) followed by evaluation of ECM production and deposition. Expression of proteins and genes corresponding to ECM were measured using Western blotting and quantitative RT‐PCR with subsequent matrix metalloproteinase (MMP) activity. Molecular mechanisms of ER activation in regulating ECM were evaluated by luciferase reporter assays for activator protein 1 (AP‐1) and NF‐κB. TNF‐α or PDGF significantly ( P < 0.001) increased ECM deposition and MMP activity in human ASM cells, which was significantly reduced with WAY treatment but not with PPT. Furthermore, TNF‐α‐ or PDGF‐induced ECM gene expression in ASM cells was significantly reduced with WAY ( P < 0.001). Moreover, WAY significantly down‐regulated the activation of NF‐κB( P < 0.001) and AP‐1 ( P < 0.01, P < 0.05) in ASM cells from asthmatics and nonasthmatics. Overall, we demonstrate differential ER signaling in controlling ECM production and deposition. Activation of ER‐β diminishes ECM deposition via suppressing the NF‐κB pathway activity and might serve as a novel target to blunt airway remodeling.—Ambhore, N. S., Kalidhindi, R. S. R., Pabelick, C. M., Hawse, J. R., Prakash, Y. S., Sathish, V. Differential estrogen‐receptor activation regulates extracellular matrix deposition in human airway smooth muscle remodeling via NF‐κB pathway. FASEB J. 33, 13935‐13950 (2019). www.fasebj.org

Asthma is defined as chronic inflammation of the airways and is characterized by airway remodeling, hyperresponsiveness, and acute bronchoconstriction of airway smooth muscle (ASM) cells. Clinical findings suggest a higher incidence and severity of asthma in adult women, indicating a concrete role of sex steroids in modulating the airway tone. Estrogen, a major female sex steroid mediates its role through estrogen receptors (ER) ERα and ERβ, which are shown to be expressed in human ASM, and their expression is upregulated in lung inflammation and asthma. Previous studies suggested rapid, nongenomic signaling of estrogen via ERs reduces intracellular calcium ([Ca2+]i), thereby promoting relaxation of ASM. However, long-term ER activation on [Ca2+]iregulation in human ASM during inflammation or in asthma is still not known. In Fura-2-loaded nonasthmatic and asthmatic human ASM cells, we found that prolonged (24 h) exposure to ERα agonist (PPT) increased [Ca2+]iresponse to histamine, whereas ERβ activation (WAY) led to decreased [Ca2+] compared with vehicle. This was further confirmed by ER overexpression and knockdown studies using various bronchoconstrictor agents. Interestingly, ERβ activation was more effective than 17β-estradiol in reducing [Ca2+]iresponses in the presence of TNF-α or IL-13, while no observable changes were noticed with PPT in the presence of either cytokine. The [Ca2+]i-reducing effects of ERβ were mediated partially via L-type calcium channel inhibition and increased Ca2+sequestration by sarcoplasmic reticulum.Overall, these data highlight the differential signaling of ERα and ERβ in ASM during inflammation. Specific ERβ activation reduces [Ca2+]iin the inflamed ASM cells and is likely to play a crucial role in regulating ASM contractility, thereby relaxing airways.

Hyperoxia exposure in premature infants increases the risk of subsequent lung diseases, such as asthma and bronchopulmonary dysplasia. Fibroblasts help maintain bronchial and alveolar integrity. Thus, understanding mechanisms by which hyperoxia influences fibroblasts is critical. Cellular senescence is increasingly recognized as important to the pathophysiology of multiple diseases. We hypothesized that clinically relevant moderate hyperoxia (<50% O2) induces senescence in developing fibroblasts. Using primary human fetal lung fibroblasts, we investigated effects of 40% O2 on senescence, endoplasmic reticulum (ER) stress, and autophagy pathways. Fibroblasts were exposed to 21% or 40% O2 for 7 days with etoposide as a positive control to induce senescence, evaluated by morphological changes, β-galactosidase activity, and DNA damage markers. Senescence-associated secretory phenotype (SASP) profile of inflammatory and profibrotic markers was further assessed. Hyperoxia decreased proliferation but increased cell size. SA-β-gal activity and DNA damage response, cell cycle arrest in G2/M phase, and marked upregulation of phosphorylated p53 and p21 were noted. Reduced autophagy was noted with hyperoxia. mRNA expression of proinflammatory and profibrotic factors (TNF-α, IL-1, IL-8, MMP3) was elevated by hyperoxia or etoposide. Hyperoxia increased several SASP factors (PAI-1, IL1-α, IL1-β, IL-6, LAP, TNF-α). The secretome of senescent fibroblasts promoted extracellular matrix formation by naïve fibroblasts. Overall, we demonstrate that moderate hyperoxia enhances senescence in primary human fetal lung fibroblasts with reduced autophagy but not enhanced ER stress. The resulting SASP is profibrotic and may contribute to abnormal repair in the lung following hyperoxia.

Airway nerves represent a mechanistically and therapeutically important aspect that requires better highlighting in the context of diseases such as asthma. Altered structure and function (plasticity) of afferent and efferent airway innervation can contribute to airway diseases. We describe established anatomy, current understanding of how plasticity occurs, and contributions of plasticity to asthma, focusing on target-derived growth factors (neurotrophins). Perspectives toward novel treatment strategies and future research are provided.

Pulmonary fibrosis is a devastating disease characterized by accumulation of activated fibroblasts and scarring in the lung. While fibroblast activation in physiological wound repair reverses spontaneously, fibroblast activation in fibrosis is aberrantly sustained. Here we identified histone 3 lysine 9 methylation (H3K9me) as a critical epigenetic modification that sustains fibroblast activation by repressing the transcription of genes essential to returning lung fibroblasts to an inactive state. We show that the histone methyltransferase G9a (EHMT2) and chromobox homolog 5 (CBX5, also known as HP1α), which deposit H3K9me marks and assemble an associated repressor complex, respectively, are essential to initiation and maintenance of fibroblast activation specifically through epigenetic repression of peroxisome proliferator–activated receptor γ coactivator 1 α gene ( PPARGC1A, encoding PGC1α). Both TGF-β and increased matrix stiffness potently inhibit PGC1α expression in lung fibroblasts through engagement of the CBX5/G9a pathway. Inhibition of the CBX5/G9a pathway in fibroblasts elevates PGC1α, attenuates TGF-β– and matrix stiffness–promoted H3K9 methylation, and reduces collagen accumulation in the lungs following bleomycin injury. Our results demonstrate that epigenetic silencing mediated by H3K9 methylation is essential for both biochemical and biomechanical fibroblast activation and that targeting this epigenetic pathway may provide therapeutic benefit by returning lung fibroblasts to quiescence.

Reactive airway diseases are significant sources of pulmonary morbidity in neonatal and pediatric patients. Supplemental oxygen exposure in premature infants contributes to airway diseases such as asthma and promotes development of airway remodeling, characterized by increased airway smooth muscle (ASM) mass and extracellular matrix (ECM) deposition. Decreased plasma membrane caveolin-1 (CAV1) expression has been implicated in airway disease and may contribute to airway remodeling and hyperreactivity. Here, we investigated the impact of clinically relevant moderate hyperoxia (50% O2) on airway remodeling and caveolar protein expression in a neonatal mouse model. Within 12 h of birth, litters of B6129SF2J mice were randomized to room air (RA) or 50% hyperoxia exposure for 7 days with or without caveolin-1 scaffolding domain peptide (CSD; caveolin-1 mimic; 10 µl, 0.25 mM daily via intraperitoneal injection) followed by 14 days of recovery in normoxia. Moderate hyperoxia significantly increased airway reactivity and decreased pulmonary compliance at 3 wk. Histologic assessment demonstrated airway wall thickening and increased ASM mass following hyperoxia. RNA from isolated ASM demonstrated significant decreases in CAV1 and cavin-1 in hyperoxia-exposed animals while cavin-3 was increased. Supplementation with intraperitoneal CSD mitigated both the physiologic and histologic changes observed with hyperoxia. Overall, these data show that moderate hyperoxia is detrimental to developing airway and may predispose to airway reactivity and remodeling. Loss of CAV1 is one mechanism through which hyperoxia produces these deleterious effects. Supplementation of CAV1 using CSD or similar analogs may represent a new therapeutic avenue for blunting hyperoxia-induced pulmonary damage in neonates.

Cellular senescence results in cell cycle arrest with secretion of cytokines, chemokines, growth factors, and remodeling proteins (senescence-associated secretory phenotype; SASP) that have autocrine and paracrine effects on the tissue microenvironment. SASP can promote remodeling, inflammation, infectious susceptibility, angiogenesis, and proliferation, while hindering tissue repair and regeneration. While the role of senescence and the contributions of senescent cells are increasingly recognized in the context of aging and a variety of disease states, relatively less is known regarding the portfolio and influences of senescent cells in normal lung growth and aging per se or in the induction or progression of lung diseases across the age spectrum such as bronchopulmonary dysplasia, asthma, chronic obstructive pulmonary disease, or pulmonary fibrosis. In this review, we introduce concepts of cellular senescence, the mechanisms involved in the induction of senescence, and the SASP portfolio that are relevant to lung cells, presenting the potential contribution of senescent cells and SASP to inflammation, hypercontractility, and remodeling/fibrosis: aspects critical to a range of lung diseases. The potential to blunt lung disease by targeting senescent cells using a novel class of drugs (senolytics) is discussed. Potential areas for future research on cellular senescence in the lung are identified.