Pabelick, Christina M.

Abstract Airway smooth muscle (ASM) regulation of airway structure and contractility is critical in fetal/neonatal physiology in health and disease. Fetal lungs experience higher Ca 2+ environment that may impact extracellular Ca 2+ ([Ca 2+ ] o ) sensing receptor (CaSR). Well‐known in the parathyroid gland, CaSR is also expressed in late embryonic lung mesenchyme. Using cells from 18‐22 week human fetal lungs, we tested the hypothesis that CaSR regulates intracellular Ca 2+ ([Ca 2+ ] i ) in fetal ASM (fASM). Compared with adult ASM, CaSR expression was higher in fASM, while fluorescence Ca 2+ imaging showed that [Ca 2+ ] i was more sensitive to altered [Ca 2+ ] o . The fASM [Ca 2+ ] i responses to histamine were also more sensitive to [Ca 2+ ] o (0–2 mM) compared with an adult, enhanced by calcimimetic R568 but blunted by calcilytic NPS2143. [Ca 2+ ] i was enhanced by endogenous CaSR agonist spermine (again higher sensitivity compared with adult). Inhibition of phospholipase C (U73122; siRNA) or inositol 1,4,5‐triphosphate receptor (Xestospongin C) blunted [Ca 2+ ] o sensitivity and R568 effects. NPS2143 potentiated U73122 effects. Store‐operated Ca 2+ entry was potentiated by R568. Traction force microscopy showed responsiveness of fASM cellular contractility to [Ca 2+ ] o and NPS2143. Separately, fASM proliferation showed sensitivity to [Ca 2+ ] o and NPS2143. These results demonstrate functional CaSR in developing ASM that modulates airway contractility and proliferation.

Abstract Supplemental O2 (hyperoxia; 30–90% O2) is a necessary intervention for premature infants, but it contributes to development of neonatal and pediatric asthma, necessitating better understanding of contributory mechanisms in hyperoxia-induced changes to airway structure and function. In adults, environmental stressors promote formation of senescent cells that secrete factors (senescence-associated secretory phenotype), which can be inflammatory and have paracrine effects that enhance chronic lung diseases. Hyperoxia-induced changes in airway structure and function are mediated in part by effects on airway smooth muscle (ASM). In the present study, using human fetal ASM cells as a model of prematurity, we ascertained the effects of clinically relevant moderate hyperoxia (40% O2) on cellular senescence. Fetal ASM exposed to 40% O2 for 7 days exhibited elevated concentrations of senescence-associated markers, including β-galactosidase; cell cycle checkpoint proteins p16, p21, and p-p53; and the DNA damage marker p-γH2A.X (phosphorylated γ-histone family member X). The combination of dasatinib and quercetin, compounds known to eliminate senescent cells (senolytics), reduced the number of hyperoxia-exposed β-galactosidase–, p21-, p16-, and p-γH2A.X–positive ASM cells. The senescence-associated secretory phenotype profile of hyperoxia-exposed cells included both profibrotic and proinflammatory mediators. Naive ASM exposed to media from hyperoxia-exposed senescent cells exhibited increased collagen and fibronectin and higher contractility. Our data show that induction of cellular senescence by hyperoxia leads to secretion of inflammatory factors and has a functional effect on naive ASM. Cellular senescence in the airway may thus contribute to pediatric airway disease in the context of sequelae of preterm birth.

ABSTRACT Recent studies have demonstrated an effect of neurotrophins, particularly brain‐derived neurotrophic factor (BDNF), on airway contractility [ via increased airway smooth muscle (ASM) intracellular calcium [Ca 2+ ] i ] and remodeling (ASM proliferation and extracellular matrix formation) in the context of airway disease. In the present study, we examined the role of BDNF in allergen‐induced airway inflammation using 2 transgenic models: 1 ) tropomyosin‐related kinase B (TrkB) conditional knockin (TrkBKI) mice allowing for inducible, reversible disruption of BDNF receptor kinase activity by administration of 1NMPP1, a PP1 derivative, and 2) smooth muscle‐specific BDNF knockout (BDNF fl/fl /SMMHCll Cre/0 ) mice. Adult mice were intranasally challenged with PBS or mixed allergen ( Altemaria alternata, Aspergillus fumigatus , house dust mite, and ovalbumin) for 4 wk. Our data show that administration of 1NMPP1 in TrkBKI mice during the 4‐wk allergen challenge blunted airway hyper‐responsiveness (AHR) and reduced fibronectin mRNA expression in ASM layers but did not reduce inflammation per se . Smooth muscle‐specific deletion of BDNF reduced AHR and blunted airway fibrosis but did not significantly alter airway inflammation. Together, our novel data indicate that TrkB signaling is a key modulator of AHR and that smooth muscle‐derived BDNF mediates these effects during allergic airway inflammation.—Britt, R. D., Jr., Thompson, M. A., Wicher, S. A., Manlove, L. J., Roesler, A., Fang, Y.‐H., Roos, C., Smith, L., Miller, J. D., Pabelick, C. M., Prakash, Y. S. Smooth muscle brain‐derived neurotrophic factor contributes to airway hyperreactivity in a mouse model of allergic asthma. FASEB J. 33, 3024–3034 (2019). www.fasebj.org

Corticosteroids (CSs) are commonly used to manage wheezing and asthma in pediatric populations. Although corticosteroids are effective in alleviating airway diseases, some children with more moderate-severe asthma phenotypes show CS resistance and exhibit significant airflow obstruction, persistent inflammation, and more frequent exacerbations. Previous studies have demonstrated that Th1 cytokines, such as TNF-α and IFN-γ, promote CS resistance in adult human airway smooth muscle (ASM). In the present study, using a human fetal ASM cell model, we tested the hypothesis that TNF-α/IFN-γ induces CS resistance. In contrast to TNF-α or IFN-γ alone, the combination of TNF-α/IFN-γ blunted the ability of fluticasone propionate (FP) to reduce expression of the chemokines CCL5 and CXCL10 despite expression of key anti-inflammatory glucocorticoid receptor target genes being largely unaffected by TNF-α/IFN-γ. Expression of the NF-κB subunit p65 and phosphorylation of Stat1 were elevated in cells treated with TNF-α/IFN-γ, an effect that remained in the presence of FP. siRNA knockdown studies demonstrated the effects of TNF-α/IFN-γ on increased p65 are mediated by Stat1, a transcription factor activated by IFN-γ. Expression of TNFAIP3, a negative regulator of NF-κB activity, was not altered by TNF-α/IFN-γ. However, the effects of TNF-α/IFN-γ were partially reduced by overexpression of TNFAIP3 but did not influence p65 expression. Together, these data suggest that IFN-γ augments the effects of TNF-α on chemokines by enhancing expression of key inflammatory pathways in the presence of CS. Interactions between TNF-α- and IFN-γ-mediated pathways may promote inflammation in asthmatic children resistant to CSs.

ABSTRACT Myofibroblasts play key roles in wound healing and pathological fibrosis. Here, we used an RNAi screen to characterize myofibroblast regulatory genes, using a high-content imaging approach to quantify α-smooth muscle actin stress fibers in cultured human fibroblasts. Screen hits were validated on physiological compliance hydrogels, and selected hits tested in primary fibroblasts from patients with idiopathic pulmonary fibrosis. Our RNAi screen led to the identification of STAT3 as an essential mediator of myofibroblast activation and function. Strikingly, we found that STAT3 phosphorylation, while responsive to exogenous ligands on both soft and stiff matrices, is innately active on a stiff matrix in a ligand/receptor-independent, but ROCK- and JAK2-dependent fashion. These results demonstrate how a cytokine-inducible signal can become persistently activated by pathological matrix stiffening. Consistent with a pivotal role for this pathway in driving persistent fibrosis, a STAT3 inhibitor attenuated murine pulmonary fibrosis when administered in a therapeutic fashion after bleomycin injury. Our results identify novel genes essential for the myofibroblast phenotype, and point to STAT3 as an important target in pulmonary fibrosis and other fibrotic diseases.

This study tested the hypothesis that the NO donor S-nitrosoglutathione (GSNO) relaxes canine tracheal smooth muscle (CTSM) in part by a cGMP-independent process that involves reversible oxidation of intracellular thiols. GSNO caused a concentration-dependent relaxation in ACh-contracted strips (EC 50 ∼1.2 μM) accompanied by a concentration-dependent increase in cytosolic cGMP concentration ([cGMP] i ). The soluble guanylate cyclase inhibitor methylene blue prevented the increase in [cGMP] i induced by 1 and 10 μM GSNO, but isometric force decreased by 10 ± 4 and 55 ± 3%, respectively. After recovery of [cGMP] i to baseline, GSNO-induced relaxation persisted during continuous ACh stimulation. Dithiothreitol caused a rapid recovery of isometric force to values similar to those obtained with ACh alone in these strips. We conclude that GSNO relaxes CTSM contracted by ACh in part by oxidation of intracellular protein thiols.

The effect of halothane on intracellular Ca2+concentration ([Ca2+]i) regulation in porcine tracheal smooth muscle cells was examined with real-time confocal microscopy. Both 1 and 2 minimum alveolar concentration (MAC) halothane increased basal [Ca2+]iwhen Ca2+influx and efflux were blocked, suggesting increased sarcoplasmic reticulum (SR) Ca2+leak and/or decreased reuptake. In β-escin-permeabilized cells, heparin inhibition of inositol 1,4,5-trisphosphate-receptor channels blunted the halothane-induced increase in [Ca2+]i. Both 1 and 2 MAC halothane decreased the frequency and amplitude of ACh-induced [Ca2+]ioscillations (which represent SR Ca2+release through ryanodine-receptor channels), abolishing oscillations in ∼20% of tracheal smooth muscle cells at 2 MAC. When Ca2+influx and efflux were blocked, halothane increased the baseline and decreased the frequency and amplitude of [Ca2+]ioscillations, inhibiting oscillations in ∼70% of cells at 2 MAC. The fall time of [Ca2+]ioscillations and the rate of fall of the [Ca2+]iresponse to caffeine were both increased by halothane. These results suggest that halothane abolishes agonist-induced [Ca2+]ioscillations by 1) depleting SR Ca2+via increased Ca2+leak through inositol 1,4,5-trisphosphate-receptor channels, 2) decreasing Ca2+release through ryanodine-receptor channels, and 3) inhibiting reuptake.

The purpose of this study was to examine whether the nitric oxide donor S-nitrosoglutathione (GSNO) relaxes canine tracheal smooth muscle (CTSM) strips by decreasing Ca 2+ sensitivity [i.e., the amount of force for a given intracellular Ca 2+ concentration ([Ca 2+ ] i )]. We further investigated whether GSNO decreases Ca 2+ sensitivity by altering the relationship between regulatory myosin light chain (rMLC) phosphorylation and [Ca 2+ ] i and the relationship between force and rMLC phosphorylation. GSNO (100 μM) relaxed intact CTSM strips contracted with 45 mM KCl by decreasing Ca 2+ sensitivity in comparison to control strips without significantly decreasing [Ca 2+ ] i . GSNO reduced the amount of rMLC phosphorylation for a given [Ca 2+ ] i but did not affect the relationship between isometric force and rMLC phosphorylation. These results show that in CTSM strips contracted with KCl, GSNO decreases Ca 2+ sensitivity by affecting the level of rMLC phosphorylation for a given [Ca 2+ ] i , suggesting that myosin light chain kinase is inhibited or that smooth muscle protein phosphatases are activated by GSNO.

In airway smooth muscle (ASM), ACh induces propagating intracellular Ca2+concentration ([Ca2+]i) oscillations (5–30 Hz). We hypothesized that, in ASM, coupling of elevations and reductions in [Ca2+]ito force generation and relaxation (excitation-contraction coupling) is slower than ACh-induced [Ca2+]ioscillations, leading to stable force generation. When we used real-time confocal imaging, the delay between elevated [Ca2+]iand contraction in intact porcine ASM cells was found to be ∼450 ms. In β-escin-permeabilized ASM strips, photolytic release of caged Ca2+resulted in force generation after ∼800 ms. When calmodulin (CaM) was added, this delay was shortened to ∼500 ms. In the presence of exogenous CaM and 100 μM Ca2+, photolytic release of caged ATP led to force generation after ∼80 ms. These results indicated significant delays due to CaM mobilization and Ca2+-CaM activation of myosin light chain kinase but much shorter delays introduced by myosin light chain kinase-induced phosphorylation of the regulatory myosin light chain MLC20and cross-bridge recruitment. This was confirmed by prior thiophosphorylation of MLC20, in which force generation occurred ∼50 ms after photolytic release of caged ATP, approximating the delay introduced by cross-bridge recruitment alone. The time required to reach maximum steady-state force was >15 s. Rapid chelation of [Ca2+]iafter photolytic release of caged diazo-2 resulted in relaxation after a delay of ∼1.2 s and 50% reduction in force after ∼57 s. We conclude that in ASM cells agonist-induced [Ca2+]ioscillations are temporally and spatially integrated during excitation-contraction coupling, resulting in stable force production.

The multiplicity of mechanisms involved in regulation of intracellular Ca2+concentration ([Ca2+]i) in smooth muscle results in both intra- and intercellular heterogeneities in [Ca2+]i. Heterogeneity in [Ca2+]iregulation is reflected by the presence of spontaneous, localized [Ca2+]itransients (Ca2+sparks) representing Ca2+release through ryanodine receptor (RyR) channels. Ca2+sparks display variable spatial Ca2+distributions with every occurrence within and across cellular regions. Individual sparks are often grouped, and fusion of sparks produces large local elevations in [Ca2+]ithat occasionally trigger propagating [Ca2+]iwaves. Ca2+sparks may modulate membrane potential and thus smooth muscle contractility. Sparks may also be the target of other regulatory factors in smooth muscle. Agonists induce propagating [Ca2+]ioscillations that originate from foci with high spark incidence and also represent Ca2+release through RyR channels. With increasing agonist concentration, the peak of regional [Ca2+]ioscillations remains relatively constant, whereas both frequency and propagation velocity increase. In contrast, the global cellular response appears as a concentration-dependent increase in peak as well as mean cellular [Ca2+]i, representing a spatial and temporal integration of the oscillations. The significance of agonist-induced [Ca2+]ioscillations lies in the establishment of a global [Ca2+]ilevel for slower Ca2+-dependent physiological processes.