Jane E. Bourke

Effects of PPARγ ligands on TGF-β1-induced epithelial-mesenchymal transition in alveolar epithelial cells

BackgroundTransforming growth factor β1 (TGF-β1)-mediated epithelial mesenchymal transition (EMT) of alveolar epithelial cells (AEC) may contribute to lung fibrosis. Since PPARγ ligands have been shown to inhibit fibroblast activation by TGF-β1, we assessed the ability of the thiazolidinediones rosiglitazone (RGZ) and ciglitazone (CGZ) to regulate TGF-β1-mediated EMT of A549 cells, assessing changes in cell morphology, and expression of cell adhesion molecules E-cadherin (epithelial cell marker) and N-cadherin (mesenchymal cell marker), and collagen 1α1 (COL1A1), CTGF and MMP-2 mRNA.MethodsSerum-deprived A549 cells (human AEC cell line) were pre-incubated with RGZ and CGZ (1 - 30 μM) in the absence or presence of the PPARγ antagonist GW9662 (10 μM) before TGFβ-1 (0.075-7.5 ng/ml) treatment for up to 72 hrs. Changes in E-cadherin, N-cadherin and phosphorylated Smad2 and Smad3 levels were analysed by Western blot, and changes in mRNA levels including COL1A1 assessed by RT-PCR.ResultsTGFβ-1 (2.5 ng/ml)-induced reductions in E-cadherin expression were associated with a loss of epithelial morphology and cell-cell contact. Concomitant increases in N-cadherin, MMP-2, CTGF and COL1A1 were evident in predominantly elongated fibroblast-like cells. Neither RGZ nor CGZ prevented TGFβ1-induced changes in cell morphology, and PPARγ-dependent inhibitory effects of both ligands on changes in E-cadherin were only evident at submaximal TGF-β1 (0.25 ng/ml). However, both RGZ and CGZ inhibited the marked elevation of N-cadherin and COL1A1 induced by TGF-β1 (2.5 ng/ml), with effects on COL1A1 prevented by GW9662. Phosphorylation of Smad2 and Smad3 by TGF-β1 was not inhibited by RGZ or CGZ.ConclusionsRGZ and CGZ inhibited profibrotic changes in TGF-β1-stimulated A549 cells independently of inhibition of Smad phosphorylation. Their inhibitory effects on changes in collagen I and E-cadherin, but not N-cadherin or CTGF, appeared to be PPARγ-dependent. Further studies are required to unravel additional mechanisms of inhibition of TGF-β1 signalling by thiazolidinediones and their implications for the contribution of EMT to lung fibrosis.

Direct and continuous detection of ATP secretion from primary monolayer cultures of bovine adrenal chromaffin cells

Abstract: A method was developed for direct and continuous detection of secretion of ATP from primary monolayer cultures of bovine adrenal chromaffin cells. ATP, which is costored with catecholamines within adrenal chromaffin cells, was released into the incubation medium, where it reacted with firefly luciferin‐luciferase producing light detected by a photomultiplier located directly below the culture well. Acetylcholine, nicotine, the Ca2+ ionophore A23187, BaCl2, and KC1 induced release of ATP. Induction of release of ATP by acetylcholine was dose dependent, with a threshold at 10‐7M and a maximum at 10‐4M. The dose‐response curve for nicotine was bell shaped, with a threshold at 10‐7M, a maximum at 10‐5M, and diminished release at higher concentrations, an observation indicative of desensitization. Investigation of the initial rates of ATP secretion revealed that 10‐4M nicotine actually induced release of ATP at a faster rate than 10‐5M nicotine. However, the rate of ATP release evoked by 10‐4M nicotine began to decline by 6 s, a result indicating the onset of receptor desensitization, whereas release induced by 10‐5M nicotine continued unabated. Induction of release of ATP by acetylcholine or nicotine was biphasic, with a rapid, initial phase of release followed by a plateau at 0.5–1.5 min and a second phase of release beginning at 1.5–2 min, reaching a maximum by 2–3 min. Comparison of the time course for ATP release evoked by 10‐4M nicotine with the time course for secretion of adrenaline and noradrenaline revealed that adrenaline was the predominant catecholamine released during the initial rapid phase of ATP release. A slight plateau for adrenaline release coincided with the plateau for ATP release. Finally, the second phase of ATP release coincided with the time at which release of noradrenaline exceeded the release of adrenaline. The initial phase of ATP release could originate primarily from adrenaline‐containing cells, whereas the second phase could occur primarily from noradrenaline‐containing cells in the monolayer culture.

Collagen remodelling by airway smooth muscle is resistant to steroids and β2-agonists

Bi-directional interactions between airway smooth muscle (ASM) and the altered extracellular matrix (ECM) may influence airway wall remodelling and ASM function in asthma. We have investigated the capacity of cultured human ASM to reorganise the structure of three-dimensional collagen gels and the effects of endothelin (ET)-1 and agents used to treat asthma. Human ASM cells were cast in type I collagen gels. Reductions in gel area over 72 h were determined in the absence and presence of ET-1 and potential inhibitors, steroids and &bgr;2-adrenoceptor agonists. Changes in gel wet weights and hydroxyproline content were measured and ASM gel morphology was examined by scanning electron microscopy. Cell density-dependent reductions in gel area were augmented by ET-1, mediated via ETA receptors. This process was not associated with ASM contraction or proliferation, but was consistent with ASM tractional remodelling and migration leading to collagen condensation rather than collagen degradation within gels. The collagen remodelling by ASM was unaffected by salbutamol and/or budesonide. This study demonstrates an additional potential role for ASM in ECM regulation and dysregulation in airways disease that is resistant to steroids and &bgr;2-adrenoceptor agonists. Therapy-resistant collagen condensation within ASM bundles may facilitate ECM-ASM interactions and contribute to increased internal airways resistance.

Relationships between adult asthma and oxidative stress markers and pH in exhaled breath condensate: a systematic review

Oxidative stress has a recognized role in the pathophysiology of asthma. Recently, interest has increased in the assessment of pH and airway oxidative stress markers. Collection of exhaled breath condensate (EBC) and quantification of biomarkers in breath samples can potentially indicate lung disease activity and help in the study of airway inflammation, and asthma severity. Levels of oxidative stress markers in the EBC have been systematically evaluated in children with asthma; however, there is no such systematic review conducted for adult asthma. A systematic review of oxidative stress markers measured in EBC of adult asthma was conducted, and studies were identified by searching MEDLINE and SCOPUS databases. Sixteen papers met the inclusion criteria. Concentrations of exhaled hydrogen ions, nitric oxide products, hydrogen peroxide and 8‐isoprostanes were generally elevated and related to lower lung function tests in adults with asthma compared to healthy subjects. Assessment of EBC markers may be a noninvasive approach to evaluate airway inflammation, exacerbations, and disease severity of asthma, and to monitor the effectiveness of anti‐inflammatory treatment regimens. Longitudinal studies, using standardized analytical techniques for EBC collection, are required to establish reference values for the interpretation of EBC markers in the context of asthma.

Emerging mediators of airway smooth muscle dysfunction in asthma.

Phenotypic changes in airway smooth muscle are integral to the pathophysiological changes that constitute asthma - namely inflammation, airway wall remodelling and bronchial hyperresponsiveness. In vitro and in vivo studies have shown that the proliferative, secretory and contractile functions of airway smooth muscle are dysfunctional in asthma. These functions can be modulated by various mediators whose levels are altered in asthma, derived from inflammatory cells or produced by airway smooth muscle itself. In this review, we describe the emerging roles of the CXC chemokines (GROs, IP-10), Th17-derived cytokines (IL-17, IL-22) and semaphorins, as well as the influence of viral infection on airway smooth muscle function, with a view to identifying new opportunities for therapeutic intervention in asthma.

Differential Effects of Allergen Challenge on Large and Small Airway Reactivity in Mice

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/TNFα 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.

PPARγ Ligands Regulate Noncontractile and Contractile Functions of Airway Smooth Muscle: Implications for Asthma Therapy

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 γ (PPARγ), a receptor upregulated in ASM in asthmatic airways, may provide a novel approach to regulate these contributions. This review summarises experimental evidence that PPARγ 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. PPARγ ligands can also regulate ASM contractility, with acute treatment eliciting relaxation of mouse trachea in vitro through a PPARγ-independent mechanism. Chronic treatment can protect against the loss of bronchodilator sensitivity to β 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 PPARγ agonists to target the noncontractile and contractile functions of ASM to improve outcomes for patients with poorly controlled asthma.

Measurement and impact of remodeling in the lung: airway neovascularization in asthma.

Expansion of the airway wall vascular compartment has recently been established as a feature of asthma involving both enlargement of existing vascular structures and the formation of new vessels (angiogenesis). Both processes are likely to occur together and are fundamental for supporting the many aspects of tissue inflammation and remodeling manifest in the clinical symptoms of airway disease. Multiple growth factors are implicated in airway angiogenesis, with vascular endothelial growth factor among the most important. Other asthma-associated stimuli, including ADAM33, environmental tobacco smoke, and rhinovirus infection, are emerging as proangiogenic regulators. Increasing attention is also focused on the complex interplay of airway wall inflammatory and structural cells, including airway smooth muscle in driving expansion of the bronchial submucosal vascular plexus in asthma. Here, we provide a brief update on recent developments in this emerging area and highlight the potential role played by airway smooth muscle.

Small-molecule-biased formyl peptide receptor agonist compound 17b protects against myocardial ischaemia-reperfusion injury in mice

Effective treatment for managing myocardial infarction (MI) remains an urgent, unmet clinical need. Formyl peptide receptors (FPR) regulate inflammation, a major contributing mechanism to cardiac injury following MI. Here we demonstrate that FPR1/FPR2-biased agonism may represent a novel therapeutic strategy for the treatment of MI. The small-molecule FPR1/FPR2 agonist, Compound 17b (Cmpd17b), exhibits a distinct signalling fingerprint to the conventional FPR1/FPR2 agonist, Compound-43 (Cmpd43). In Chinese hamster ovary (CHO) cells stably transfected with human FPR1 or FPR2, Compd17b is biased away from potentially detrimental FPR1/2-mediated calcium mobilization, but retains the pro-survival signalling, ERK1/2 and Akt phosphorylation, relative to Compd43. The pathological importance of the biased agonism of Cmpd17b is demonstrable as superior cardioprotection in both in vitro (cardiomyocytes and cardiofibroblasts) and MI injury in mice in vivo. These findings reveal new insights for development of small molecule FPR agonists with an improved cardioprotective profile for treating MI.

Novel Small Airway Bronchodilator Responses to Rosiglitazone in Mouse Lung Slices

There is a need to identify novel agents that elicit small airway relaxation when β2-adrenoceptor agonists become ineffective in difficult-to-treat asthma. Because chronic treatment with the synthetic peroxisome proliferator activated receptor (PPAR)γ 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 β-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 β-adrenoceptor antagonist propranolol and was enhanced by ALB. RGZ-induced relaxation was mimicked by other synthetic PPARγ agonists but not by the putative endogenous agonist 15-deoxy-PGJ2 and was not prevented by the PPARγ antagonist GW9662. To induce airway relaxation, RGZ inhibited the amplitude and frequency of MCh-induced Ca(2+) oscillations of airway smooth muscle cells (ASMCs). In addition, RGZ reduced MCh-induced Ca(2+) sensitivity of the ASMCs. Collectively, these findings demonstrate that acute bronchodilator responses induced by RGZ are PPARγ independent, additive with ALB, and occur by the inhibition of ASMC Ca(2+) signaling and Ca(2+) sensitivity. Because RGZ continues to elicit relaxation when β-adrenoceptor agonists have a limited effect, RGZ or related compounds may have potential as bronchodilators for the treatment of difficult asthma.