We have employed a simple and robust noninvasive method of continuous in vivo long-term bromodeoxyuridine (BrdU) labeling to analyze lung mesenchymal stromal cell turnover in adult mice in the steady state. Mathematical modeling of BrdU uptake in flow cytometrically sorted CD45(neg)CD31(neg)Sca-1(pos) lung cells following long-term feeding of BrdU to mice in their drinking water reveals that lung mesenchymal stromal cells cycle continuously throughout life. Analysis of BrdU incorporation during long-term feeding and during chasing (delabeling) following replacement of BrdU-water with normal water shows that the CD45(neg)CD31(neg)Sca-1(pos) lung mesenchymal stromal cell compartment turns over at a rate of similar to 2.26% per day with a time to half-cycled of 44 days, an estimated cell proliferation rate of 0.004/day, and a cell death rate of 0.018/day.

There is a need to identify novel agents that elicit small airway relaxation when beta(2)-adrenoceptor agonists become ineffective in difficult-to-treat asthma. Because chronic treatment with the synthetic peroxisome proliferator activated receptor (PPAR) g agonist rosiglitazone (RGZ) inhibits airway hyperresponsiveness in mouse models of allergic airways disease, we tested the hypothesis that RGZ causes acute airway relaxation by measuring changes in small airway size in mouse lung slices. Whereas the beta-adrenoceptor agonists albuterol (ALB) and isoproterenol induced partial airway relaxation, RGZ reversed submaximal and maximal contraction to methacholine (MCh) and was similarly effective after precontraction with serotonin or endothelin-1. Concentration-dependent relaxation to RGZ was not altered by the b-adrenoceptor antagonist propranolol and was enhanced by ALB. RGZ-induced relaxation was mimicked by other synthetic PPAR gamma agonists but not by the putative endogenous agonist 15-deoxy-PGJ(2) and was not prevented by the PPARg antagonist GW9662. To induce airway relaxation, RGZ inhibited the amplitude and frequency of MCh-induced Ca2+ oscillations of airway smooth muscle cells (ASMCs). In addition, RGZ reduced MCh-induced Ca2+ sensitivity of the ASMCs. Collectively, these findings demonstrate that acute bronchodilator responses induced by RGZ are PPARg independent, additive with ALB, and occur by the inhibition of ASMC Ca2+ signaling and Ca2+ sensitivity. Because RGZ continues to elicit relaxation when b-adrenoceptor agonists have a limited effect, RGZ or related compounds may have potential as bronchodilators for the treatment of difficult asthma.

In asthma, basic fibroblast growth factor (FGF-2) plays an important (patho) physiological role. This study examines the effects of FGF-2 on the transforming growth factor-beta (TGF-beta)-stimulated differentiation of airway smooth muscle (ASM) cells in vitro. The differentiation of human ASM cells after incubation with TGF-beta (100 pM) and/or FGF-2 (300 pM) for 48 hours was assessed by increases in contractile protein expression, actin-cytoskeleton reorganization, enhancements in cell stiffness, and collagen remodeling. FGF-2 inhibited TGF-beta-stimulated increases in transgelin (SM22) and calponin gene expression (n = 15, P < 0.01) in an extracellular signal-regulated kinase 1/2 (ERK1/2) signal transduction-dependent manner. The abundance of ordered alpha-smooth muscle actin (alpha-SMA) filaments formed in the presence of TGF-beta were also reduced by FGF-2, as was the ratio of F-actin to G-actin (n = 8, P < 0.01). Furthermore, FGF-2 attenuated TGF-beta-stimulated increases in ASM cell stiffness and the ASM-mediated contraction of lattices, composed of collagen fibrils (n = 5, P < 0.01). However, the TGF-beta-stimulated production of IL-6 was not influenced by FGF-2 (n = 4, P > 0.05), suggesting that FGF-2 antagonism is selective for the regulation of ASM cell contractile protein expression, organization, and function. Another mitogen, thrombin (0.3 U ml(-1)), exerted no effect on TGF-beta-regulated contractile protein expression (n = 8, P > 0.05), alpha-SMA organization, or the ratio of F-actin to G-actin (n = 4, P > 0.05), suggesting that the inhibitory effect of FGF-2 is dissociated from its mitogenic actions. The addition of FGF-2, 24 hours after TGF-beta treatment, still reduced contractile protein expression, even when the TGF-beta-receptor kinase inhibitor, SB431542 (10 mu M), was added 1 hour before FGF-2. We conclude that the ASM cell differentiation promoted by TGF-beta is antagonized by FGF-2. A better understanding of the mechanism of action for FGF-2 is necessary to develop a strategy for therapeutic exploitation in the treatment of asthma.

Background: We have previously shown that transforming growth factor-beta (TGF-beta) impairs glucocorticoid (GC) function in pulmonary epithelial cell-lines. However, the signalling cascade leading to this impairment is unknown. In the present study, we provide the first evidence that TGF-beta impairs GC action in differentiated primary air-liquid interface (ALI) human bronchial epithelial cells (HBECs). Using the BEAS-2B bronchial epithelial cell line, we also present a systematic examination of the known pathways activated by TGF-beta, in order to ascertain the molecular mechanism through which TGF-beta impairs epithelial GC action.Methods: GC transactivation was measured using a Glucocorticoid Response Element (GRE)-Secreted embryonic alkaline phosphatase (SEAP) reporter and measuring GC-inducible gene expression by qRT-PCR. GC transrepression was measured by examining GC regulation of pro-inflammatory mediators. TGF-beta signalling pathways were investigated using siRNA and small molecule kinase inhibitors. GRa level, phosphorylation and sub-cellular localisation were determined by western blotting, immunocytochemistry and localisation of GRa-Yellow Fluorescent Protein (YFP). Data are presented as the mean +/- SEM for n independent experiments in cell lines, or for experiments on primary HBEC cells from n individual donors. All data were statistically analysed using GraphPad Prism 5.0 (Graphpad, San Diego, CA). In most cases, two-way analyses of variance (ANOVA) with Bonferroni post-hoc tests were used to analyse the data. In all cases, P < 0.05 was considered to be statistically significant.Results: TGF-beta impaired Glucocorticoid Response Element (GRE) activation and the GC induction of several anti-inflammatory genes, but did not broadly impair the regulation of pro-inflammatory gene expression in A549 and BEAS-2B cell lines. TGF-beta-impairment of GC transactivation was also observed in differentiated primary HBECs. The TGF-beta receptor (ALK5) inhibitor SB431541 fully prevented the GC transactivation impairment in the BEAS-2B cell line. However, neither inhibitors of the known downstream non-canonical signalling pathways, nor knocking down Smad4 by siRNA prevented the TGF-beta impairment of GC activity.Conclusions: Our results indicate that TGF-beta profoundly impairs GC transactivation in bronchial epithelial cells through activating ALK5, but not through known non-canonical pathways, nor through Smad4-dependent signalling, suggesting that TGF-beta may impair GC action through a novel non-canonical signalling mechanism.

Background: Current therapy for relieving bronchoconstriction may be ineffective in severe asthma, particularly in the small airways. The aim of this study was to further characterise responses to the recently identified novel bronchodilators rosiglitazone (RGZ) and chloroquine (CQ) under conditions where beta-adrenoceptor agonist efficacy was limited or impaired in mouse small airways within lung slices.Methods: Relaxation to RGZ and CQ was assessed following submaximal methacholine (MCh) pre-contraction, in slices treated overnight with either RGZ, CQ or albuterol (ALB) ( to induce beta-adrenoceptor desensitization), and in slices treated with caffeine/ryanodine in which contraction is associated with increases in Ca2+ sensitivity in the absence of contractile agonist-induced Ca2+ oscillations. Furthermore, the effects of RGZ, CQ, ALB and isoproterenol (ISO) on the initiation and development of methacholine-induced contraction were also compared.Results: RGZ and CQ, but not ALB or ISO, elicited complete relaxation with increasing MCh pre-contraction and maintained their potency and efficacy following beta-adrenoceptor desensitization. RGZ, CQ and ALB maintained efficacy following overnight incubation with RGZ or CQ. Relaxation responses to all dilators were generally maintained but delayed after caffeine/ryanodine. Pre-treatment with RGZ, but not CQ, ALB or ISO, reduced MCh potency.Conclusions: This study demonstrates the superior effectiveness of RGZ in comparison to CQ and beta-adrenoceptor agonists as a dilator of mouse small airways. Further investigation of the mechanisms underlying the relatively greater efficacy of RGZ under these conditions are warranted and should be extended to include studies in human asthmatic airways.

Background: LysoTracker Green DND-26 is a fluorescent dye that stains acidic compartments in live cells and has been shown to selectively accumulate in lamellar bodies in alveolar type II (AT2) cells in the lung. The aim of this study was to determine whether the accumulation of LysoTracker in lamellar bodies can be used to isolate viable AT2 cells by flow cytometry and track their differentiation in live-cell culture by microscopy.Methods: Mouse lung cells were sorted on the basis of CD45(neg)CD31(neg)EpCAM(pos)LysoTracker(pos) expression and characterized by immunostaining for SP-C and cultured in a three-dimensional epithelial colony-forming unit (CFU-Epi) assay. To track AT2 cell differentiation, lung epithelial stem and progenitor cells were cultured in a CFU-Epi assay with LysoTracker-supplemented media.Results: The purity of sorted AT2 cells as determined by SP-C staining was 97.4% and viability was 85.3%. LysoTracker(pos) AT2 cells generated SP-C-pos alveolar epithelial cell colonies in culture, and when added to the CFU-Epi culture medium, LysoTracker marked the differentiation of stem/progenitor-derived AT2 cells.Conclusions: This study describes a novel method for isolating AT2 cells from mouse lungs. The high purity and viability of cells attained by this method, makes them suitable for functional analysis in vitro. The application of LysoTracker to live cell cultures will allow better assessment of the cellular and molecular mechanisms that regulate AT2 cell differentiation.

The relative contributions of large and small airways to hyperresponsiveness in asthma have yet to be fully assessed. This study used a mouse model of chronic allergic airways disease to induce inflammation and remodelling and determine whether in vivo hyperresponsiveness to methacholine is consistent with in vitro reactivity of trachea and small airways. Balb/C mice were sensitised (days 0, 14) and challenged (3 times/week, 6 weeks) with ovalbumin. Airway reactivity was compared with saline-challenged controls in vivo assessing whole lung resistance, and in vitro measuring the force of tracheal contraction and the magnitude/rate of small airway narrowing within lung slices. Increased airway inflammation, epithelial remodelling and fibrosis were evident following allergen challenge. In vivo hyperresponsiveness to methacholine was maintained in isolated trachea. In contrast, methacholine induced slower narrowing, with reduced potency in small airways compared to controls. In vitro incubation with IL-1/TNF alpha did not alter reactivity. The hyporesponsiveness to methacholine in small airways within lung slices following chronic ovalbumin challenge was unexpected, given hyperresponsiveness to the same agonist both in vivo and in vitro in tracheal preparations. This finding may reflect the altered interactions of small airways with surrounding parenchymal tissue after allergen challenge to oppose airway narrowing and closure.

In asthma, the increase in airway smooth muscle (ASM) can contribute to inflammation, airway wall remodeling and airway hyperresponsiveness (AHR). Targetting peroxisome proliferator-activated receptor gamma (PPAR gamma.), a receptor upregulated in ASM in asthmatic airways, may provide a novel approach to regulate these contributions. This review summarises experimental evidence that PPAR. ligands, such as rosiglitazone (RGZ) and pioglitazone (PGZ), inhibit proliferation and inflammatory cytokine production from ASM in vitro. In addition, inhaled administration of these ligands reduces inflammatory cell infiltration and airway remodelling in mouse models of allergen-induced airways disease. PPAR gamma ligands can also regulate ASM contractility, with acute treatment eliciting relaxation of mouse trachea in vitro through a PPAR gamma-independent mechanism. Chronic treatment can protect against the loss of bronchodilator sensitivity to beta(2)-adrenoceptor agonists and inhibit the development of AHR associated with exposure to nicotine in utero or following allergen challenge. Of particular interest, a small clinical trial has shown that oral RGZ treatment improves lung function in smokers with asthma, a group that is generally unresponsive to conventional steroid treatment. These combined findings support further investigation of the potential for PPAR. agonists to target the noncontractile and contractile functions of ASM to improve outcomes for patients with poorly controlled asthma.