Michelle Freeman

Cigarette smoke-induced mitochondrial fragmentation and dysfunction in human airway smooth muscle

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.

Calcium-sensing receptor antagonists abrogate airway hyperresponsiveness and inflammation in allergic asthma

Calcilytics reduce airway hyperresponsiveness and inflammation and may represent effective asthma therapeutics. Calcilytics may help asthmatics breathe easier Calcium may help to build strong bones. However, Yarova et al. now show that extracellular calcium may contribute to inflammation and airway hyperresponsiveness in allergic asthma. They show that elevated extracellular calcium can activate airway smooth muscle cells through the calcium-sensing receptor (CaSR). Asthmatic patients express higher levels of CaSR in their airways than do healthy individuals, as does a mouse model of allergic asthma. Indeed, extracellular calcium and other asthma-associated activators of CaSR increased airway hyperreactivity. What’s more, calcilytics—CaSR antagonists—can prevent these effects both in vitro and in vivo, supporting clinical testing of these drugs for asthmatics. Airway hyperresponsiveness and inflammation are fundamental hallmarks of allergic asthma that are accompanied by increases in certain polycations, such as eosinophil cationic protein. Levels of these cations in body fluids correlate with asthma severity. We show that polycations and elevated extracellular calcium activate the human recombinant and native calcium-sensing receptor (CaSR), leading to intracellular calcium mobilization, cyclic adenosine monophosphate breakdown, and p38 mitogen-activated protein kinase phosphorylation in airway smooth muscle (ASM) cells. These effects can be prevented by CaSR antagonists, termed calcilytics. Moreover, asthmatic patients and allergen-sensitized mice expressed more CaSR in ASMs than did their healthy counterparts. Indeed, polycations induced hyperreactivity in mouse bronchi, and this effect was prevented by calcilytics and absent in mice with CaSR ablation from ASM. Calcilytics also reduced airway hyperresponsiveness and inflammation in allergen-sensitized mice in vivo. These data show that a functional CaSR is up-regulated in asthmatic ASM and targeted by locally produced polycations to induce hyperresponsiveness and inflammation. Thus, calcilytics may represent effective asthma therapeutics.

Sex Steroids Influence Brain-Derived Neurotropic Factor Secretion From Human Airway Smooth Muscle Cells.

Brain derived neurotropic factor (BDNF) is emerging as an important player in airway inflammation, remodeling, and hyperreactivity. Separately, there is increasing evidence that sex hormones contribute to pathophysiology in the lung. BDNF and sex steroid signaling are thought to be intricately linked in the brain. There is currently little information on BDNF and sex steroid interactions in the airway but is relevant to understanding growth factor signaling in the context of asthma in men versus women. In this study, we assessed the effect of sex steroids on BDNF expression and secretion in human airway smooth muscle (ASM). Human ASM was treated with estrogen (E2) or testosterone (T, 10 nM each) and intracellular BDNF and secreted BDNF measured. E2 and T significantly reduced secretion of BDNF; effects prevented by estrogen and androgen receptor inhibitor, ICI 182,780 (1 μM), and flutamide (10 μM), respectively. Interestingly, no significant changes were observed in intracellular BDNF mRNA or protein expression. High affinity BDNF receptor, TrkB, was not altered by E2 or T. E2 (but not T) significantly increased intracellular cyclic AMP levels. Notably, Epac1 and Epac2 expression were significantly reduced by E2 and T. Furthermore, SNARE complex protein SNAP25 was decreased. Overall, these novel data suggest that physiologically relevant concentrations of E2 or T inhibit BDNF secretion in human ASM, suggesting a potential interaction of sex steroids with BDNF in the airway that is different from brain. The relevance of sex steroid–BDNF interactions may lie in their overall contribution to airway diseases such as asthma. J. Cell. Physiol. 231: 1586–1592, 2016.

Hyperinsulinemia Adversely Affects Lung Structure and Function

There is limited knowledge regarding the consequences of hyperinsulinemia on the lung. Given the increasing prevalence of obesity, insulin resistance, and epidemiological associations with asthma, this is a critical lacuna, more so with inhaled insulin on the horizon. Here, we demonstrate that insulin can adversely affect respiratory health. Insulin treatment (1 μg/ml) significantly (P < 0.05) increased the proliferation of primary human airway smooth muscle (ASM) cells and induced collagen release. Additionally, ASM cells showed a significant increase in calcium response and mitochondrial respiration upon insulin exposure. Mice administered intranasal insulin showed increased collagen deposition in the lungs as well as a significant increase in airway hyperresponsiveness. PI3K/Akt mediated activation of β-catenin, a positive regulator of epithelial-mesenchymal transition and fibrosis, was observed in the lungs of insulin-treated mice and lung cells. Our data suggests that hyperinsulinemia may have adverse effects on airway structure and function. Insulin-induced activation of β-catenin in lung tissue and the contractile effects on ASM cells may be causally related to the development of asthma-like phenotype.

Mechanisms of Cigarette Smoke Effects on Human Airway Smooth Muscle.

Cigarette smoke contributes to or exacerbates airway diseases such as asthma and COPD, where airway hyperresponsiveness and airway smooth muscle (ASM) proliferation are key features. While factors such as inflammation contribute to asthma in part by enhancing agonist-induced intracellular Ca2+ ([Ca2+]i) responses of ASM, the mechanisms by which cigarette smoke affect ASM are still under investigation. In the present study, we tested the hypothesis that cigarette smoke enhances the expression and function of Ca2+ regulatory proteins leading to increased store operated Ca2+ entry (SOCE) and cell proliferation. Using isolated human ASM (hASM) cells, incubated in the presence and absence cigarette smoke extract (CSE) we determined ([Ca2+]i) responses and expression of relevant proteins as well as ASM proliferation, reactive oxidant species (ROS) and cytokine generation. CSE enhanced [Ca2+]i responses to agonist and SOCE: effects mediated by increased expression of TRPC3, CD38, STIM1, and/or Orai1, evident by attenuation of CSE effects when siRNAs against these proteins were used, particularly Orai1. CSE also increased hASM ROS generation and cytokine secretion. In addition, we found in the airways of patients with long-term smoking history, TRPC3 and CD38 expression were significantly increased compared to life-long never-smokers, supporting the role of these proteins in smoking effects. Finally, CSE enhanced hASM proliferation, an effect confirmed by upregulation of PCNA and Cyclin E. These results support a critical role for Ca2+ regulatory proteins and enhanced SOCE to alter airway structure and function in smoking-related airway disease.

Cigarette Smoke and Estrogen Signaling in Human Airway Smooth Muscle

Aims: Cigarette smoke (CS) in active smokers and second-hand smoke exposure exacerbate respiratory disorders such as asthma and chronic bronchitis. While women are known to experience a more asthmatic response to CS than emphysema in men, there is limited information on the mechanisms of CS-induced airway dysfunction. We hypothesize that CS interferes with a normal (protective) bronchodilatory role of estrogens, thus worsening airway contractility. Methods: We tested effects of cigarette smoke extract (CSE) on 17β-estradiol (E2) signaling in enzymatically-dissociated bronchial airway smooth muscle (ASM) obtained from lung samples of non-smoking female patients undergoing thoracic surgery. Results: In fura-2 loaded ASM cells, CSE increased intracellular calcium ([Ca2+]i) responses to 10µM histamine. Acute exposure to physiological concentrations of E2 decreased [Ca2+]i responses. However, in 24h exposed CSE cells, although expression of estrogen receptors was increased, the effect of E2 on [Ca2+]i was blunted. Acute E2 exposure also decreased store-operated Ca2+ entry and inhibited stromal interaction molecule 1 (STIM1) phosphorylation: effects blunted by CSE. Acute exposure to E2 increased cAMP, but less so in 24h CSE-exposed cells. 24h CSE exposure increased S-nitrosylation of ERα. Furthermore, 24h CSE-exposed bronchial rings showed increased bronchoconstrictor agonist responses that were not reduced as effectively by E2 compared to non-CSE controls. Conclusion: These data suggest that CS induces dysregulation of estrogen signaling in ASM, which could contribute to increased airway contractility in women exposed to CS.

Vitamin D Reduces Inflammation-induced Contractility and Remodeling of Asthmatic Human Airway Smooth Muscle

BACKGROUND Although multiple clinical studies have found an association between vitamin D (Vit D) deficiency and asthma, a recent clinical study suggested lack of therapeutic effect of Vit D supplementation. Nonetheless, the mechanisms by which Vit D influences airway structure and function in the context of inflammation and asthma remains undefined. In this regard, Vit D effects on airway smooth muscle (ASM) are important, given the role of this cell type in the hypercontractility and remodeling. We assessed the mechanisms by which Vit D modulates the enhancing effects of proinflammatory cytokines tumor necrosis factor-α (TNF-α) and IL-13 on intracellular Ca(2+) ([Ca(2+)]i) levels and remodeling in nonasthmatic versus asthmatic human ASM. METHODS Human ASM was enzymatically isolated from surgical lung specimens of patients with clinically defined mild to moderate asthma versus no asthma. Cells were treated with 10 ng/ml TNF-α and 50 ng/ml IL-13 in the presence or absence of 100 nM calcitriol. MEASUREMENTS AND MAIN RESULTS Interestingly, Vit D receptor (VDR) and retinoic X receptor-α levels were maintained, even increased, in subjects with asthma when treated with TNF-α and IL-13. Compared with untreated cells, calcitriol blunted the heightened effect of TNF-α on [Ca(2+)]i response to histamine in ASM. Calcitriol particularly blunted TNF-α and IL-13 effects on collagen and fibronectin deposition, especially in asthmatic ASM. Calcitriol stimulated VDR/retinoic X receptor dimerization and VDR activity even in subjects with asthma and with IL-13, highlighting retained functionality. Expression of Class I histone deacetylases 1-3 (HDAC) and overall HDAC activity were lower in IL-13-exposed ASM, but calcitriol enhanced HDAC expression/activity. CONCLUSIONS In asthmatic ASM, Vit D functionality is maintained, allowing calcitriol to reduce the procontractile and proremodeling effects of inflammatory cytokines, particularly IL-13, which is relevant to asthma. These findings highlight a potential role for Vit D in asthma pathogenesis, particularly in the context of airway structure and functional changes early in disease.

Author response to letter to editor: Hyperinsulinemia adversely affects lung structure and function

to the editor: We appreciate the interest shown by Wolff et al. ([17][1]) regarding our recent publication in the American Journal of Physiology Lung Cellular and Molecular Physiology ([11][2]). We acknowledge the convenience of an inhaled insulin formulation, the extensive safety data required by

RNAi screening identifies a mechanosensitive ROCK-JAK2- STAT3 network central to myofibroblast activation

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. Summary: Myofibroblasts are key drivers of fibrosis. Here, RNAi screening is used to identify novel regulators of myofibroblast activation, and a matrix-stiffness-dependent STAT3 activation pathway is identified.