Judith C.W. Mak

Protective effects of a glucocorticoid on downregulation of pulmonary beta 2-adrenergic receptors in vivo.

We investigated the in vivo effects of a glucocorticoid on beta-agonist-induced downregulation of beta 1- and beta 2-adrenergic receptors (determined by [125I]iodocyanopindolol binding), mRNA expression (assessed by Northern blotting), and gene transcription (using nuclear run-on assays) in rat lung. Dexamethasone (Dex) (0.2 mg/kg/d, days 1-8) increased beta 1- and beta 2-receptor numbers by 70 and 69% above control, respectively, but did not change their mRNA expression. Isoproterenol (Iso) (0.96 mg/kg/d, days 2-8) decreased beta 1- and beta 2-receptor numbers by 48 and 51%, respectively, and also reduced mRNA expression by 69 and 57%, respectively. The combination of Dex and Iso resulted in no net change in beta 2-receptor number and its mRNA expression, although there was a significant reduction in beta 1-receptor number and mRNA expression. The mapping of beta 1- and beta 2-receptors by receptor autoradiography confirmed these findings over alveoli, epithelium, endothelium, and airway and vascular smooth muscle. We also measured the activation of the transcription factor, cyclic AMP response element binding protein (CREB) using an electrophoretic mobility shift assay. CREB-like DNA-binding activity was decreased after Iso treatment but this decrease was prevented after treatment with Dex. Nuclear run-on assays revealed that the transcription rate of the beta 1-receptor gene did not alter after Dex treatment, but was reduced after Iso treatment. The transcription rate of the beta 2-receptor gene was increased after Dex treatment by approximately twofold, but there was no change after Iso treatment. We conclude that glucocorticoids can prevent homologous downregulation of beta 2-receptor number and mRNA expression at the transcriptional level without affecting beta 1-receptors and that the transcription factor CREB may be involved in this phenomenon. Such an effect may have clinical implications for preventing the development of tolerance to beta 2-agonists in asthmatic patients treated with beta-agonist bronchodilators.

Inhaled fluticasone in bronchiectasis: a 12 month study

Background: The clinical efficacy of inhaled corticosteroid (ICS) treatment has not been evaluated in bronchiectasis, despite the presence of chronic airway inflammation. Methods: After three consecutive weekly visits, 86 patients were randomised to receive either fluticasone 500 μg twice daily (n = 43, 23F, mean (SD) age 57.7 (14.4) years) or matched placebo (n = 43, 34F, 59.2 (14.2) years) and reviewed regularly for 52 weeks in a double blind fashion. Results: 35 and 38 patients in the fluticasone and placebo groups completed the study. Significantly more patients on ICS than on placebo showed improvement in 24 hour sputum volume (OR 2.5, 95% CI 1.1 to 6.0, p = 0.03) but not in exacerbation frequency, forced expiratory volume in 1 second, forced vital capacity, or sputum purulence score. Significantly more patients with Pseudomonas aeruginosa infection receiving fluticasone showed improvement in 24 hour sputum volume (OR 13.5, 95% CI 1.8 to 100.2, p = 0.03) and exacerbation frequency (OR 13.3, 95% CI 1.8 to 100.2, p = 0.01) than those given placebo. Logistic regression models revealed a significantly better response in sputum volume with fluticasone treatment than with placebo among subgroups of patients with 24 hour sputum volume <30 ml (p = 0.04), exacerbation frequency ⩽2/year (p = 0.04), and sputum purulence score >5 (p = 0.03). Conclusions: ICS treatment is beneficial to patients with bronchiectasis, particularly those with P aerurginosa infection.

Tiotropium bromide (Ba 679 BR), a novel long-acting muscarinic antagonist for the treatment of obstructive airways disease

Tiotropium bromide (Ba 679 BR) is a novel potent and long-lasting muscarinic antagonist that has been developed for the treatment of chronic obstructive airways disease (COPD). Binding studies with [3H]tiotropium bromide in human lung have confirmed that this is a potent muscarinic antagonist with equal affinity for M1-, M2- and M3-receptors and is approximately 10-fold more potent than ipratropium bromide. Tiotropium bromide dissociates very slowly from lung muscarinic receptors compared with ipratropium bromide. In vitro tiotropium bromide has a potent inhibitory effect against cholinergic nerve-induced contraction of guinea-pig and human airways, that has a slower onset than atropine or ipratropium bromide. After washout, however, tiotropium bromide dissociates extremely slowly compared with the dissociation of atropine and ipratropium bromide. Measurement of acetylcholine (ACh) release from guinea-pig trachea shows that tiotropium bromide, ipratropium bromide and atropine all increase ACh release on neural stimulation and that this effect is washed out equally quickly for the three antagonists. This confirms binding studies to transfected human muscarinic receptors which suggested that tiotropium bromide dissociates slowly from M3-receptors (on airway smooth muscle) but rapidly from M2 autoreceptors (on cholinergic nerve terminals). Clinical studies with inhaled tiotropium bromide confirm that it is a potent and long-lasting bronchodilator in COPD and asthma. Furthermore, it protects against cholinergic bronchoconstriction for > 24 h. This suggests that tiotropium bromide will be a useful bronchodilator, particularly in patients with COPD, and may be suitable for daily dosing. The selectivity for M3- over M2-receptors may also confer a clinical advantage.

C-Reactive Protein Is Associated With Obstructive Sleep Apnea Independent of Visceral Obesity

BACKGROUND Obstructive sleep apnea (OSA) is associated with adverse cardiovascular outcomes. C-reactive protein (CRP) predicts atherosclerotic complications. Our study evaluates whether OSA is associated with an elevated CRP level, after elimination of known confounders including visceral obesity. METHODS Men without significant chronic medical illness, regular medications, or illness in the preceding 4 weeks were enrolled. Subjects with morbid obesity, newly detected high BP, or fasting glucose were excluded. They underwent polysomnography and MRI of abdomen to quantify visceral fat volume. High-sensitivity CRP levels were measured. RESULTS 111 men with mean body mass index (BMI) 26.3 +/- 3.8 kg/m(2) were evaluated. After adjustment for age, smoking, BMI, waist circumference, and sleep efficiency, CRP correlated positively with the apnea-hypopnea index (AHI) [r = 0.35, p < 0.001], duration of O(2) saturation < 90% (r = 0.29, p = 0.002), and arousal index (r = 0.32, p = 0.001), and it correlated negatively with minimal O(2) saturation (r = -0.29, p = 0.002). These correlations were consistent when adjustment was made for MRI visceral fat volume instead of waist circumference. In the regression model, significant predictors of CRP included AHI, waist circumference, and triglycerides (adjusted R(2), 0.33, p = 0.001, p = 0.002, p = 0.018, respectively). Among the 111 subjects, 32 subjects with no or mild OSA (AHI < 15 events/h) were matched with 32 subjects with moderate-to-severe OSA (AHI > or = 15 events/h) in MRI visceral fat volume. CRP was higher in subjects with moderate-to-severe OSA (median, 1.32; 0.45 to 2.34 mg/L) when compared to subjects with no or mild OSA (median, 0.54; 0.25 to 0.89 mg/L; p = 0.001). CONCLUSIONS In healthy middle-aged men, elevated CRP level is associated with OSA independent of visceral obesity.

Mitochondrial transfer of induced pluripotent stem cell-derived mesenchymal stem cells to airway epithelial cells attenuates cigarette smoke-induced damage.

Transplantation of mesenchymal stem cells (MSCs) holds great promise in the repair of cigarette smoke (CS)-induced lung damage in chronic obstructive pulmonary disease (COPD). Because CS leads to mitochondrial dysfunction, we aimed to investigate the potential benefit of mitochondrial transfer from human-induced pluripotent stem cell-derived MSCs (iPSC-MSCs) to CS-exposed airway epithelial cells in vitro and in vivo. Rats were exposed to 4% CS for 1 hour daily for 56 days. At Days 29 and, human iPSC-MSCs or adult bone marrow-derived MSCs (BM-MSCs) were administered intravenously to CS-exposed rats. CS-exposed rats exhibited severe alveolar destruction with a higher mean linear intercept (Lm) than sham air-exposed rats (P < 0.001) that was attenuated in the presence of iPSC-MSCs or BM-MSCs (P < 0.01). The attenuation of Lm value and the severity of fibrosis was greater in the iPSC-MSC-treated group than in the BM-MSC-treated group (P < 0.05). This might have contributed to the novel observation of mitochondrial transfer from MSCs to rat airway epithelial cells in lung sections exposed to CS. In vitro studies further revealed that transfer of mitochondria from iPSC-MSCs to bronchial epithelial cells (BEAS-2B) was more effective than from BM-MSCs, with preservation of adenosine triphosphate contents. This distinct mitochondrial transfer occurred via the formation of tunneling nanotubes. Inhibition of tunneling nanotube formation blocked mitochondrial transfer. Our findings indicate a higher mitochondrial transfer capacity of iPSC-MSCs than BM-MSCs to rescue CS-induced mitochondrial damage. iPSC-MSCs may thus hold promise for the development of cell therapy in COPD.

Obesity, obstructive sleep apnoea and metabolic syndrome

OSA is increasingly recognized as a major health problem in developed countries. Obesity is the most common risk factor in OSA and hence, the prevalence of OSA is undoubtedly rising given the epidemic of obesity. Recent data also suggest that OSA is highly associated with the metabolic syndrome, and it is postulated that OSA contributes to cardiometabolic dysfunction, and subsequently vasculopathy.

Mechanisms of impaired beta-adrenoceptor-induced airway relaxation by interleukin-1beta in vivo in the rat.

We studied the in vivo mechanism of beta-adrenergic receptor (beta-AR) hyporesponsiveness induced by intratracheal instillation of interleukin-1beta (IL-1beta, 500 U) in Brown-Norway rats. Tracheal and bronchial smooth muscle responses were measured under isometric conditions ex vivo. Contractile responses to electrical field stimulation and to carbachol were not altered, but maximal relaxation induced by isoproterenol (10(-6)-10(-5) M) was significantly reduced 24 h after IL-1beta treatment in tracheal tissues and to a lesser extent, in the main bronchi. Radioligand binding using [125I]iodocyanopindolol revealed a 32+/-7% reduction in beta-ARs in lung tissues from IL-1beta-treated rats, without any significant changes in beta2-AR mRNA level measured by Northern blot analysis. Autoradiographic studies also showed significant reduction in beta2-AR in the airways. Isoproterenol-stimulated cyclic AMP accumulation was reduced by IL-1beta at 24 h in trachea and lung tissues. Pertussis toxin reversed this hyporesponsiveness to isoproterenol but not to forskolin in lung tissues. Western blot analysis revealed an IL-1beta-induced increase in Gi(alpha) protein expression. Thus, IL-1beta induces an attenuation of beta-AR-induced airway relaxation through mechanisms involving a reduction in beta-ARs, an increase in Gi(alpha) subunit, and a defect in adenylyl cyclase activity.

Localization of β2-adrenoceptor messenger RNA in human and rat lung using in situ hybridization: correlation with receptor autoradiography

We have used in situ hybridization to study the localization of mRNA encoding the beta 2-adrenoceptor in tissue sections of the human and rat lung and compared this with the distribution of beta 2-receptor binding sites using receptor autoradiography. To localize beta 2-receptor mRNA, a [32P]labeled antisense RNA probe (riboprobe) was generated from human or rat beta 2-receptor cDNA. A similar distribution of beta 2-receptor mRNA was identified in both species. The highest intensity of beta 2-receptor mRNA was detected in smooth muscle of small airways, airway epithelium and pulmonary blood vessels. Lower intensity of beta 2-receptor mRNA was identified in smooth muscle of large airways, and alveolar epithelium (presumably type I and type II pneumocytes). No significant hybridization signal was detected in interstitial tissue. The specificity of the hybridization signal was confirmed with a sense probe (having identical sequence to the mRNA) and preincubation with RNase A, and by Northern blot analysis which revealed a single band of mRNA of 2.2 kb. There was a correspondence between mRNA localization and the distribution of beta 2-receptors visualized by [125I]iodocyanopindolol autoradiographically in the presence of CGP 20712 (a beta 1-selective antagonist). However, alveolar walls that showed a high beta 2-receptor density had relatively low levels of mRNA. This cellular heterogeneity may reflect differences in RNA stability or transcription rate in different lung cells. This approach opens up new options in the investigation of the regulation of pulmonary beta 2-receptor gene expression in health and disease.

Autoradiographic localization of calcitonin gene-related peptide (CGRP) binding sites in human and guinea pig lung

125I-Human calcitonin gene-related peptide (hCGRP) binding sites were localized in human and guinea pig lungs by an autoradiographic method. Scatchard analysis of saturation experiments from slide-mounted sections of guinea pig lung displayed specific 125I-hCGRP binding sites with a dissociation constant (Kd) of 0.72 +/- 0.05 nM (mean +/- S.E.M., n = 3) and a maximal number of binding sites (Bmax) of 133.4 +/- 5.6 fmol/mg protein. In both human and guinea pig lung, autoradiography revealed that CGRP binding sites were widely distributed, with particularly dense labeling over bronchial and pulmonary blood vessels of all sizes and alveolar walls. Airway smooth muscle and epithelium of large airways was sparsely labeled but no labeling was found over submucosal glands. This localization corresponds well to the reported pattern of CGRP-like immunoreactive innervation. The findings of localization of CGRP binding sites on bronchial and pulmonary blood vessels indicate that CGRP may be important in the regulation of airway and pulmonary blood flow.

Analysis of the dissociated steroid RU24858 does not exclude a role for inducible genes in the anti-inflammatory actions of glucocorticoids.

Although repression of inflammatory gene expression makes glucocorticoids powerful anti-inflammatory agents, side effects limit usage and drive the search for improved glucocorticoid receptor (GR) ligands. In A549 pulmonary cells, dexamethasone and the prototypical dissociated ligand RU24858 (Mol Endocrinol 11:1245-1255, 1997) repress interleukin (IL)-1β-induced expression of cyclooxygenase (COX)-2 and IL-8. Although RU24858 is a weaker GR ligand, both glucocorticoids showed similar efficacies on transrepression of nuclear factor κB (NF-κB)-dependent transcription, whereas RU24858 yielded less than 12% of the response to dexamethasone on a classic glucocorticoid response element (GRE) reporter (transactivation). Modest NF-κB-dependent transrepression (∼40%), along with analysis of IL-8 transcription rate and the accumulation of unspliced nuclear RNA, indicates that transrepression does not fully account for the repression of genes such as IL-8. This was confirmed by the finding that mRNA degradation is increased by both dexamethasone and RU24858. Analysis of IL-1β-induced steady-state mRNA levels for IL-8 and COX-2 show that dexamethasone- and RU24858-dependent repression of these genes is attenuated by inhibitors of transcription and protein synthesis. Because similar effects were observed with respect to COX-2 and IL-8 protein expression, we conclude that glucocorticoid-dependent gene expression is necessary for repression by both glucocorticoids. Despite RU24858 being defective at classic GRE-dependent transactivation, both dexamethasone and RU24858 induced the expression of potentially anti-inflammatory genes and metabolic genes, suggesting the importance of nontraditional glucocorticoid-dependent gene expression. Thus, classic transactivation- and transrepressionbased screens for anti-inflammatory “dissociated” GR ligands may be flawed because they may not reflect the effects on real glucocorticoid-inducible genes.