Reynold A. Panettieri

Sphingosine 1-phosphate modulates human airway smooth muscle cell functions that promote inflammation and airway remodeling in asthma

Asthma is characterized by airway inflammation, remodeling, and hyperresponsiveness to contractile stimuli that promote airway constriction and wheezing. Here we present evidence that sphingosine 1‐phosphate (SPP) is a potentially important inflammatory mediator implicated in the pathogenesis of airway inflammation and asthma. SPP levels were elevated in the airways of asthmatic (but not control) subjects following segmental antigen challenge, and this increase was correlated with a concomitant increase in airway inflammation. Because human airway smooth muscle (ASM) cells expressed EDG receptors for SPP (EDG‐1, ‐3, ‐5, and ‐6), we examined whether SPP may play a role in airway inflammation and remodeling, by affecting ASM cell growth, contraction, and cytokine secretion. SPP is mitogenic and augments EGF‐ and thrombin‐induced DNA proliferation by increasing G1/S progression. SPP increased phosphoinositide turnover and intracellular calcium mobilization, the acute signaling events that affect ASM contraction. By modulating adenylate cyclase activity and cAMP accumulation, SPP had potent effects on cytokine secretion. Although SPP inhibited TNF‐α–induced RANTES release, it induced substantial IL‐6 secretion alone and augmented production of IL‐6 induced by TNF‐α. These studies are the first to associate SPP with airway inflammation and to identify SPP as an effective regulator of ASM growth, contraction and synthetic functions.

Asthma outcomes: Biomarkers

BACKGROUND Measurement of biomarkers has been incorporated within clinical research studies of asthma to characterize the population and associate the disease with environmental and therapeutic effects. OBJECTIVE National Institutes of Health institutes and federal agencies convened an expert group to propose which biomarkers should be assessed as standardized asthma outcomes in future clinical research studies. METHODS We conducted a comprehensive search of the literature to identify studies that developed and/or tested asthma biomarkers. We identified biomarkers relevant to the underlying disease process progression and response to treatment. We classified the biomarkers as either core (required in future studies), supplemental (used according to study aims and standardized), or emerging (requiring validation and standardization). This work was discussed at an National Institutes of Health-organized workshop convened in March 2010 and finalized in September 2011. RESULTS Ten measures were identified; only 1, multiallergen screening to define atopy, is recommended as a core asthma outcome. Complete blood counts to measure total eosinophils, fractional exhaled nitric oxide (Feno), sputum eosinophils, urinary leukotrienes, and total and allergen-specific IgE are recommended as supplemental measures. Measurement of sputum polymorphonuclear leukocytes and other analytes, cortisol measures, airway imaging, breath markers, and system-wide studies (eg, genomics, proteomics) are considered as emerging outcome measures. CONCLUSION The working group participants propose the use of multiallergen screening in all asthma clinical trials to characterize study populations with respect to atopic status. Blood, sputum, and urine specimens should be stored in biobanks, and standard procedures should be developed to harmonize sample collection for clinical trial biorepositories.

CD38/cyclic ADP-ribose-mediated Ca2+ signaling contributes to airway smooth muscle hyper-responsiveness

We previously demonstrated that cyclic ADP‐ribose (cADPR) elicits Ca2+ release in airway smooth muscle (ASM) cells through ryanodine receptor channels. CD38 is a cell surface protein that catalyzes the synthesis and degradation of cADPR. In inflammatory diseases such as asthma, augmented Ca2+ responses and Ca2+ sensitivity contribute to increased ASM contractility in response to agonists. In this study, we investigated the regulation of CD38 expression and the role of cADPR‐mediated Ca2+ release in airway inflammation. Human ASM cells in culture between the second and fifth passages were exposed to tumor necrosis factor α (TNF‐α), interleukin 1β, or interferon γ, or bovine serum albumin (controls). CD38 expression was measured by reverse transcriptase‐polymerase chain reaction (RT‐PCR), real‐time PCR, and Western blot analysis, and ADP‐ribosyl cyclase activity was assayed with nicotinamide guanine dinucleotide as the substrate. Ca2+ responses to acetylcholine, bradykinin, and thrombin were measured in fura‐2AM‐loaded cells by fluorescence microscopy. Cytokines caused significant augmentation of CD38 expression, ADP‐ribosyl cyclase activity, and Ca2+ responses to the agonists, compared with the control. TNF‐α effects were greater than those of the other two cytokines. The cADPR antagonist 8‐bromo‐cADPR attenuated the Ca2+ responses to the agonists in control and cytokine‐treated cells, with the magnitude of inhibition correlating with the level of CD38. This study provides the first demonstration of a role for CD38‐cADPR signaling in a model of inflammatory airway disease.

Wnt signaling regulates smooth muscle precursor development in the mouse lung via a tenascin C/PDGFR pathway

Paracrine signaling from lung epithelium to the surrounding mesenchyme is important for lung SMC development and function and is a contributing factor in an array of pulmonary diseases such as bronchopulmonary dysplasia, pulmonary hypertension, and asthma. Wnt7b, which is exclusively expressed in the lung epithelium, is important for lung vascular smooth muscle integrity, but the underlying mechanism by which Wnt signaling regulates lung SMC development is unclear. In this report, we have demonstrated that Wnt7b regulates a program of mesenchymal differentiation in the mouse lung that is essential for SMC development. Genetic loss-of-function studies showed that Wnt7b and beta-catenin were required for expression of Pdgfralpha and Pdgfrbeta and proliferation in pulmonary SMC precursors. In contrast, gain-of-function studies showed that activation of Wnt signaling increased the expression of both Pdgfralpha and Pdgfrbeta as well as the proliferation of SMC precursors. We further showed that the effect on Pdgfr expression was, in part, mediated by direct transcriptional regulation of the ECM protein tenascin C (Tnc), which was necessary and sufficient for Pdgfralpha/beta expression in lung explants. Moreover, this pathway was highly upregulated in a mouse model of asthma and in lung tissue from patients with pulmonary hypertension. Together, these data define a Wnt/Tnc/Pdgfr signaling axis that is critical for smooth muscle development and disease progression in the lung.

Benralizumab for chronic obstructive pulmonary disease and sputum eosinophilia: a randomised, double-blind, placebo-controlled, phase 2a study

BACKGROUND Chronic obstructive pulmonary disease (COPD) is associated with eosinophilic airway inflammation in 10-20% of patients. Benralizumab, an anti-interleukin-5 receptor α monoclonal antibody, depletes blood and sputum eosinophils. We aimed to establish whether benralizumab reduces acute exacerbations of COPD in patients with eosinophilia and COPD. METHODS We did this randomised, double-blind, placebo-controlled, phase 2a study between Nov 18, 2010, and July 13, 2013, at 26 sites in the UK, Poland, Germany, Canada, the USA, Denmark, and Spain. Adults aged 40-85 years, with moderate-to-severe COPD, at least one acute exacerbation of COPD, and a sputum eosinophil count of 3·0% or more within the previous year, were randomly assigned (1:1) via computer-generated permuted block randomisation (block size of four), with an interactive voice or web-response system, to receive placebo or 100 mg benralizumab subcutaneously, every 4 weeks (three doses), then every 8 weeks (five doses) over 48 weeks. Study site personnel included in study assessments, participants, and data analysts, were masked to treatment allocation. The primary endpoint was the annualised rate of acute exacerbations of COPD at week 56, defined as the number of acute exacerbations divided by total duration of person-year follow-up. Secondary and exploratory endpoints included COPD-specific Saint Georges Respiratory Questionnaire (SGRQ-C), Chronic Respiratory Questionnaire self-administered standardised format (CRQ-SAS), pre-bronchodilator forced expiratory volume in 1 second (FEV1), and safety. We did a prespecified subgroup analysis by baseline blood eosinophil count. Analyses were by intention to treat and per-protocol. This trial is registered with ClinicalTrials.gov, number NCT01227278. FINDINGS We randomly assigned 101 patients to receive placebo (n=50) or benralizumab (n=51), of whom 88 (87%) patients completed the study. Six patients who completed the study were excluded from the per-protocol population because of major protocol violations; the per-protocol population thus included 82 patients. Benralizumab did not reduce the annualised rate of acute exacerbations of COPD compared with placebo in the per-protocol population, with rates of 0·95 (0·68-1·29; n=40) versus 0·92 (0·67-1·25; n=42). Mean pre-bronchodilator FEV1 change from baseline to week 56 was -0·06 L (SD 0·24) with placebo, and 0·13 L (0·41) with benralizumab (p=0·014). Numerical, albeit non-significant, improvement in acute exacerbations of COPD, SGRQ-C, CRQ-SAS, and FEV1 were greater in benralizumab-treated patients with baseline blood eosinophil concentrations of 200 cells per μL or more or 300 cells per μL or more. Incidence of treatment-emergent adverse events was similar between the two groups, with the most common events being respiratory disorders (31 [62%] of 50 patients given placebo vs 32 [63%] of 51 given benralizumab) and infections (28 [56%] vs 27 [53%]). A higher incidence of serious treatment-emergent adverse events were recorded in patients in the benralizumab group than in those in the placebo group (14 vs nine patients), although none of these events were considered by the investigator to be benralizumab related. INTERPRETATION Compared with placebo, benralizumab did not reduce the rate of acute exacerbations of COPD. However, the results of prespecified subgroup analysis support further investigation of benralizumab in patients with COPD and eosinophilia. FUNDING MedImmune.

IL-13 enhances agonist-evoked calcium signals and contractile responses in airway smooth muscle.

Growing evidence suggests that interleukin (IL)‐13, a Th2‐type cytokine, plays a critical role in the development of bronchial hyper‐responsiveness (BHR), an essential feature of asthma, although the underlying mechanisms remain unknown. In the present study, we investigated whether IL‐13 directly affects airway smooth muscle (ASM) function. In murine tracheal rings, IL‐13 (100 ng ml−1, 24 h) significantly increased both the carbachol‐ and KCl‐induced maximal force generation without affecting ASM sensitivity. In cultured human ASM cells, IL‐13 (50 ng ml−1, 24 h) also augmented cytosolic calcium levels to bradykinin, histamine and carbachol by 60, 35 and 26%, respectively. The present study demonstrates that IL‐13 may promote BHR by directly modulating ASM contractility, an effect that may be due to enhanced G protein‐coupled receptor (GPCR)‐associated calcium signaling.

MAPK superfamily activation in human airway smooth muscle: mitogenesis requires prolonged p42/p44 activation.

Asthma is frequently associated with abnormal airway smooth muscle (ASM) growth that may contribute to airway narrowing and hyperresponsiveness to contractile agents. Although numerous hormones and cytokines have been shown to induce human ASM (HASM) proliferation, the cellular and molecular mechanisms underlying HASM hyperplasia are largely unknown. Here we characterize the roles of the mitogen-activated protein kinase (MAPK) superfamily [p42/p44 MAPK, c-Jun amino-terminal kinase/stress-activated protein kinase (JNK/SAPK), and p38] in mediating hormone- and cytokine-induced HASM proliferation. Significant enhancement of [3H]thymidine incorporation in HASM cultures was observed only by treatment with agents (epidermal growth factor, platelet-derived growth factor, thrombin, and phorbol 12-myristate 13-acetate) that promoted a strong and sustained activation of p42/p44 MAPK. Significant activation of the JNK/SAPK and p38 pathways was only observed on stimulation with interleukin (IL)-1β and tumor necrosis factor-α, agents that did not appreciably stimulate HASM proliferation. Two different inhibitors of MAPK/extracellular signal-regulated kinase kinase (MEK), PD-98059 and U-0126, inhibited mitogen-induced [3H]thymidine incorporation in a manner consistent with their ability to inhibit p42/p44 activation. Elk-1 and activator protein-1 reporter activation by mitogens was similarly inhibited by inhibition of MEK, suggesting a linkage between p42/p44 activation, transcription factor activation, and HASM proliferation. These findings establish a fundamental role for p42/p44 activation in regulating HASM proliferation and provide insight into species-specific differences observed among studies in ASM mitogenesis.

Endothelin‐1‐induced potentiation of human airway smooth muscle proliferation: an ETA receptor‐mediated phenomenon

1 . In this study the mitogenic effects in human cultured tracheal smooth muscle cells of endothelin‐1 (ET‐1), ET‐3, and sarafotoxin S6c (S6c), the ETB receptor‐selective agonist, were explored either alone or in combination with the potent mitogen, epidermal growth factor (EGF). 2 . In confluent, growth‐arrested human airway smooth, neither ET‐1 (0.01 nM‐1μm) nor ET‐3 (0.001 nM‐1 μm) or S6c (0.01 nM‐1 μm) induced cell proliferation, as assessed by [3H]‐thymidine incorporation. In contrast, EGF (1.6 pM − 16 nM) produced concentration‐dependent stimulation of DNA synthesis (EC50 of about 0.06 nM). The maximum increase of about 60 fold above control, elicited by 16nM EGF, was similar to that obtained with 10% foetal bovine serum (FBS). EGF (0.16‐16 nM) also produced a concentration‐dependent increase in cell counts, whereas ET‐1 (1 − 100 nM) was without effect on this index of mitogenesis. 3 . ET‐1 (1–100 nM) potentiated EGF‐induced proliferation of human tracheal smooth muscle cells. For example, ET‐1 (100 nM), which alone was without significant effect, increased by 3.0 to 3.5 fold the mitogenic influence of EGF (0.16 nM). The potentiating effect of ET‐1 on EGF‐induced proliferation was antagonized by BQ‐123 (3 μm), the ETA receptor antagonist, but was unaffected by the ETB receptor antagonist BQ‐788 (10 μm). 4 . Neither ET‐3 (1 − 100 nM) nor S6c (1 − 100 nM) influenced the mitogenic effects of EGF (0.16‐1.6 nM). 5 . [125I]‐ET‐1 binding studies revealed that on average the ratio of ETA to ETB receptors in human cultured tracheal smooth muscle cells was 35:65 (±3; n=4), confirming the predominance of the ETB receptor subtype in human airway smooth muscle. 6 . These data indicate that ET‐1 alone does not induce significant human airway smooth muscle cell proliferation. However, it potently potentiated mitogenesis induced by EGF, apparently via an ETA receptor‐mediated mechanism. These findings suggest that ET‐1, a mediator detected in increased amounts in patients with acute asthma, may potentiate the proliferative effects of mitogens and contribute to the airway smooth muscle hyperplasia associated with chronic severe asthma.

Phosphatidylinositol 3-kinase mediates mitogen-induced human airway smooth muscle cell proliferation.

Hypertrophy and hyperplasia of airway smooth muscle (ASM) are important pathological features that contribute to airflow obstruction in chronic severe asthma. Despite considerable research effort, the cellular mechanisms that modulate ASM growth remain unknown. Recent evidence suggests that mitogen-induced activation of phosphoinositide (PI)-specific phospholipase C (PLC) and PI-dependent calcium mobilization are neither sufficient nor necessary to stimulate human ASM proliferation. In this study, we identify phosphatidylinositol (PtdIns) 3-kinase as a key regulator of human ASM proliferation. Pretreatment of human ASM with the PtdIns 3-kinase inhibitors wortmannin and LY-294002 significantly reduced thrombin- and epidermal growth factor (EGF)-induced DNA synthesis (IC(50) approximately 10 nM and approximately 3 microM, respectively). In separate experiments, wortmannin and LY-294002 markedly inhibited PtdIns 3-kinase and 70-kDa S6 protein kinase (pp70(S6k)) activation induced by stimulation of human ASM cells with EGF and thrombin but had no effect on EGF- and thrombin-induced p42/p44 mitogen-activated protein kinase (MAPK) activation. The specificity of wortmannin and LY-294002 was further suggested by the demonstrated inability of these compounds to alter thrombin-induced calcium transients, total PI hydrolysis, or basal cAMP levels. Transient expression of constitutively active PtdIns 3-kinase (p110*) activated pp70(S6k), whereas a dominant-negative PtdIns 3-kinase (Deltap85) blocked EGF- and thrombin-stimulated pp70(S6k) activity. Collectively, these data suggest that activation of PtdIns 3-kinase is required for the mitogenic effect of EGF and thrombin in human ASM cells. Further investigation of the role of PtdIns 3-kinase may offer new therapeutic approaches in the treatment of diseases characterized by smooth muscle cell hyperplasia such as asthma and chronic bronchitis.Hypertrophy and hyperplasia of airway smooth muscle (ASM) are important pathological features that contribute to airflow obstruction in chronic severe asthma. Despite considerable research effort, the cellular mechanisms that modulate ASM growth remain unknown. Recent evidence suggests that mitogen-induced activation of phosphoinositide (PI)-specific phospholipase C (PLC) and PI-dependent calcium mobilization are neither sufficient nor necessary to stimulate human ASM proliferation. In this study, we identify phosphatidylinositol (PtdIns) 3-kinase as a key regulator of human ASM proliferation. Pretreatment of human ASM with the PtdIns 3-kinase inhibitors wortmannin and LY-294002 significantly reduced thrombin- and epidermal growth factor (EGF)-induced DNA synthesis (IC50 ∼10 nM and ∼3 μM, respectively). In separate experiments, wortmannin and LY-294002 markedly inhibited PtdIns 3-kinase and 70-kDa S6 protein kinase (pp70S6k) activation induced by stimulation of human ASM cells with EGF and thrombin but had no effect on EGF- and thrombin-induced p42/p44 mitogen-activated protein kinase (MAPK) activation. The specificity of wortmannin and LY-294002 was further suggested by the demonstrated inability of these compounds to alter thrombin-induced calcium transients, total PI hydrolysis, or basal cAMP levels. Transient expression of constitutively active PtdIns 3-kinase (p110*) activated pp70S6k, whereas a dominant-negative PtdIns 3-kinase (Δp85) blocked EGF- and thrombin-stimulated pp70S6kactivity. Collectively, these data suggest that activation of PtdIns 3-kinase is required for the mitogenic effect of EGF and thrombin in human ASM cells. Further investigation of the role of PtdIns 3-kinase may offer new therapeutic approaches in the treatment of diseases characterized by smooth muscle cell hyperplasia such as asthma and chronic bronchitis.

Vitamin D inhibits growth of human airway smooth muscle cells through growth factor‐induced phosphorylation of retinoblastoma protein and checkpoint kinase 1

Background and purpose:  Airway remodelling in asthma is manifested, in part, as increased airway smooth muscle (ASM) mass, reflecting myocyte proliferation. We hypothesized that calcitriol, a secosteroidal vitamin D receptor (VDR) modulator, would inhibit growth factor‐induced myocyte proliferation.