Prakash, Y S

Asthma in the pediatric population remains a significant contributor to morbidity and increasing healthcare costs. Vitamin D3 insufficiency and deficiency have been associated with development of asthma. Recent studies in models of adult airway diseases suggest that the bioactive Vitamin D3 metabolite, calcitriol (1,25‐dihydroxyvitamin D3; 1,25(OH)2D3), modulates responses to inflammation; however, this concept has not been explored in developing airways in the context of pediatric asthma. We used human fetal airway smooth muscle (ASM) cells as a model of the early postnatal airway to explore how calcitriol modulates remodeling induced by pro‐inflammatory cytokines. Cells were pre‐treated with calcitriol and then exposed to TNFα or TGFβ for up to 72 h. Matrix metalloproteinase (MMP) activity, production of extracellular matrix (ECM), and cell proliferation were assessed. Calcitriol attenuated TNFα enhancement of MMP‐9 expression and activity. Additionally, calcitriol attenuated TNFα and TGFβ‐induced collagen III expression and deposition, and separately, inhibited proliferation of fetal ASM cells induced by either inflammatory mediator. Analysis of signaling pathways suggested that calcitriol effects in fetal ASM involve ERK signaling, but not other major inflammatory pathways. Overall, our data demonstrate that calcitriol can blunt multiple effects of TNFα and TGFβ in developing airway, and point to a potentially novel approach to alleviating structural changes in inflammatory airway diseases of childhood. J. Cell. Physiol. 230: 1189–1198, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company

Abstract Plasma membrane Ca2+ influx, especially store-operated Ca2+ entry triggered by sarcoplasmic reticulum (SR) Ca2+ release, is a key component of intracellular calcium concentration ([Ca2+]i) regulation in airway smooth muscle (ASM). Agonist-induced Ca2+ oscillations in ASM that involve both influx and SR mechanisms have been previously demonstrated. In nonexcitable cells, [Ca2+]i oscillations involve Ca2+ influx via arachidonic acid (AA) –stimulated channels, which show similarities to store-operated Ca2+ entry, although their molecular identity remains undetermined. Little is known about AA-regulated Ca2+ channels or their regulation in ASM. In enzymatically dissociated human ASM cells loaded with the Ca2+ indicator, fura-2, AA (1–10 μM) triggered [Ca2+]i oscillations that were inhibited by removal of extracellular Ca2+. Other fatty acids, such as the diacylglycerol analog, 1-oleoyl-2-acetyl-SN-glycerol, oleic acid, and palmitic acid (10 μM each), failed to elicit similar [Ca2+]i responses. Preincubation with LaCl3 (1 μM or 1 mM) inhibited AA-induced oscillations. Inhibition of receptor-operated channels (SKF96,365 [10 μM]), lipoxygenase (zileuton [10 μM]), or cyclooxygenase (indomethacin [10 μM]) did not affect oscillation parameters. Inhibition of SR Ca2+ release (ryanodine [10 μM] or inositol 1,4,5-trisphosphate receptor inhibitor, xestospongin C [1 μM]) decreased [Ca2+]i oscillation frequency and amplitude. Small interfering RNA against caveolin-1, stromal interaction molecule 1, or Orai3 (20 nM each) reduced the frequency and amplitude of AA-induced [Ca2+]i oscillations. In ASM cells derived from individuals with asthma, AA increased oscillation amplitude, but not frequency. These results are highly suggestive of a novel AA-mediated Ca2+–regulatory mechanism in human ASM, reminiscent of agonist-induced oscillations. Given the role of AA in ASM intracellular signaling, especially with inflammation, AA-regulated Ca2+ channels could potentially contribute to increased [Ca2+]i in diseases such asthma.

Wheezing is a major long-term respiratory morbidity in preterm infants with and without bronchopulmonary dysplasia. We hypothesized that mild vs. severe hyperoxic exposure in neonatal mice differentially affects airway smooth muscle hypertrophy and resultant airway reactivity. Newborn mice were exposed to 7 days of mild (40% oxygen) or severe (70% oxygen) hyperoxia vs. room air controls. Respiratory system resistance (Rrs), compliance (Crs), and airway reactivity were measured 14 days after oxygen exposure ended under ketamine/xylazine anesthesia. Baseline Rrs increased and Crs decreased in both treatment groups. Methacholine challenge dose dependently increased Rrs and decreased Crs in 40% oxygen-exposed mice, whereas Rrs and Crs responses were similar between 70% oxygen-exposed and normoxic controls. Airway smooth muscle thickness was increased in 40%- but not 70%-exposed mice, whereas collagen increased and both alveolar number and radial alveolar counts decreased after 40% and 70% oxygen. These data indicate that severity of hyperoxia may differentially affect structural and functional changes in the developing mouse airway that contribute to longer-term hyperreactivity. These findings may be important to our understanding of the complex role of neonatal supplemental oxygen therapy in postnatal development of airway responsiveness.

The balance between mitochondrial fission and fusion is crucial for mitochondria to perform its normal cellular functions. We hypothesized that cigarette smoke (CS) disrupts this balance and enhances mitochondrial dysfunction in the airway. In nonasthmatic human airway smooth muscle (ASM) cells, CS extract (CSE) induced mitochondrial fragmentation and damages their networked morphology in a concentration-dependent fashion, via increased expression of mitochondrial fission protein dynamin-related protein 1 (Drp1) and decreased fusion protein mitofusin (Mfn) 2. CSE effects on Drp1 vs. Mfn2 and mitochondrial network morphology involved reactive oxygen species (ROS), activation of extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt), protein kinase C (PKC) and proteasome pathways, as well as transcriptional regulation via factors such as NF-κB and nuclear erythroid 2-related factor 2. Inhibiting Drp1 prevented CSE effects on mitochondrial networks and ROS generation, whereas blocking Mfn2 had the opposite, detrimental effect. In ASM from asmatic patients, mitochondria exhibited substantial morphological defects at baseline and showed increased Drp1 but decreased Mfn2 expression, with exacerbating effects of CSE. Overall, these results highlight the importance of mitochondrial networks and their regulation in the context of cellular changes induced by insults such as inflammation (as in asthma) or CS. Altered mitochondrial fission/fusion proteins have a further potential to influence parameters such as ROS and cell proliferation and apoptosis relevant to airway diseases.

Nanomedicine is a rapidly evolving field with high potential for developing novel research, diagnosis, and/or therapeutic approaches for lung diseases. However, for engineered nanomaterials to reach their true potential, there are still a number of unanswered questions regarding nanomaterial vs. tissue properties that dictate lung cellular uptake, distribution, and intracellular effects, and particle vs. tissue factors that determine toxicity vs. beneficial effects in the lung. Some of these key questions are highlighted in this Perspectives. Addressing these important issues will help improve nanoparticle design and enhance enthusiasm for more widespread use of nanotechnology in pulmonary medicine.

Abstract Idiopathic pulmonary fibrosis (IPF) is a fatal disorder resulting from the progressive remodeling of lungs, with no known effective treatment. Although transforming growth factor (TGF)-β has a well-established role in lung fibrosis, clinical experience with neutralizing antibodies to TGF-β has been disappointing, and strategies to directly suppress TGF-β1 secretion are needed. In this study we used a combination of in silico, in vitro, and in vivo approaches to identify microRNAs involved in TGF-β1 regulation and to validate the role of miR-326 in pulmonary fibrosis.We show that hsa-miR-326 regulates TGF-β1 expression and that hsa-miR-326 levels are inversely correlated to TGF-β1 protein levels in multiple human cell lines. The increase in TGF-β1 expression during the progression of bleomycin-induced lung fibrosis in mice was associated with loss of mmu-miR-326. Restoration of mmu-miR-326 levels by intranasal delivery of miR-326 mimics was sufficient to inhibit TGF-β1 expression and attenuate the fibrotic response. Moreover, human IPF lung specimens had markedly diminished miR-326 expression as compared with nonfibrotic lungs. Additional targets of miR-326 controlling TGF-β signaling and fibrosis-related pathways were identified, and miR-326 was found to down-regulate profibrotic genes, such as Ets1, Smad3, and matrix metalloproteinase 9, whereas it up-regulates antifibrotic genes, such as Smad7. Our results suggest for the first time that miR-326 plays a key role in regulating TGF-β1 expression and other profibrotic genes and could be useful in developing better therapeutic strategies for alleviating lung fibrosis.

It is now established that airway smooth muscle (ASM) has roles in determining airway structure and function, well beyond that as the major contractile element. Indeed, changes in ASM function are central to the manifestation of allergic, inflammatory, and fibrotic airway diseases in both children and adults, as well as to airway responses to local and environmental exposures. Emerging evidence points to novel signaling mechanisms within ASM cells of different species that serve to control diverse features, including 1) [Ca2+]icontractility and relaxation, 2) cell proliferation and apoptosis, 3) production and modulation of extracellular components, and 4) release of pro- vs. anti-inflammatory mediators and factors that regulate immunity as well as the function of other airway cell types, such as epithelium, fibroblasts, and nerves. These diverse effects of ASM “activity” result in modulation of bronchoconstriction vs. bronchodilation relevant to airway hyperresponsiveness, airway thickening, and fibrosis that influence compliance. This perspective highlights recent discoveries that reveal the central role of ASM in this regard and helps set the stage for future research toward understanding the pathways regulating ASM and, in turn, the influence of ASM on airway structure and function. Such exploration is key to development of novel therapeutic strategies that influence the pathophysiology of diseases such as asthma, chronic obstructive pulmonary disease, and pulmonary fibrosis.

Obesity, metabolic syndrome, and asthma are all rapidly increasing globally. Substantial emerging evidence suggests that these three conditions are epidemiologically and mechanistically linked. Since the link between obesity and asthma appears to extend beyond mechanical pulmonary disadvantage, molecular understanding is necessary. Insulin resistance is a strong, independent risk factor for asthma development, but it is unknown whether a direct effect of insulin on the lung is involved. This review summarizes current knowledge regarding the effect of insulin on cellular components of the lung and highlights the molecular consequences of insulin-related metabolic signaling cascades that could adversely affect lung structure and function. Examples include airway smooth muscle proliferation and contractility and regulatory signaling networks that are associated with asthma. These aspects of insulin signaling provide mechanistic insight into the clinical evidence for the links between obesity, metabolic syndrome, and airway diseases, setting the stage for novel therapeutic avenues targeting these conditions.

Abstract Inflammation elevates intracellular Ca2+ ([Ca2+]i) concentrations in airway smooth muscle (ASM). Store-operated Ca2+ entry (SOCE) is an important source of [Ca2+]i mediated by stromal interaction molecule–1 (STIM1), a sarcoplasmic reticulum (SR) protein. In transducing SR Ca2+ depletion, STIM1 aggregates to form puncta, thereby activating SOCE via interactions with a Ca2+ release-activated Ca2+ channel protein (Orai1) in the plasma membrane. We hypothesized that STIM1 aggregation is enhanced by inflammatory cytokines, thereby augmenting SOCE in human ASM cells. We used real-time fluorescence microscopic imaging to assess the dynamics of STIM1 aggregation and SOCE after exposure to TNF-α or IL-13 in ASM cells overexpressing yellow fluorescent protein–tagged wild-type STIM1 (WT-STIM1) and STIM1 mutants lacking the Ca2+-sensing EF-hand (STIM1-D76A), or lacking the cytoplasmic membrane binding site (STIM1ΔK). STIM1 aggregation was analyzed by monitoring puncta size during the SR Ca2+ depletion induced by cyclopiazonic acid (CPA). We found that puncta size was increased in cells expressing WT-STIM1 after CPA. However, STIM1-D76A constitutively formed puncta, whereas STIM1ΔK failed to form puncta. Furthermore, cytokines increased basal WT-STIM1 puncta size, and the SOCE triggered by SR Ca2+ depletion was increased in cells expressing WT-STIM1 or STIM1-D76A. Meanwhile, SOCE in cells expressing STIM1ΔK and STIM1 short, interfering RNA (siRNA) was decreased. Similarly, in cells overexpressing STIM1, the siRNA knockdown of Orai1 blunted SOCE. However, exposure to cytokines increased SOCE in all cells, increased basal [Ca2+]i, and decreased SR Ca2+ content. These data suggest that cytokines induce a constitutive increase in STIM1 aggregation that contributes to enhanced SOCE in human ASM after inflammation. Such effects of inflammation on STIM1 aggregations may contribute to airway hyperresponsiveness.