Esteban J. Morcillo

Investigation into the role of phosphodiesterase IV in bronchorelaxation, including studies with human bronchus

1 We have investigated the role of cyclic nucleotide phosphodiesterase IV (PDEIV) in the relaxation of human bronchus and guinea‐pig trachea in vitro and in guinea‐pigs in vivo. 2 Functional studies showed that the selective PDE IV inhibitors, rolipram and denbufylline, relaxed human and guinea‐pig preparations in vitro. 3 Two clinically used xanthine non‐selective PDE inhibitors, theophylline and pentoxifylline, were also effective in these preparations, but were much less potent than the selective agents used. 4 The rank order of potency for the four PDE inhibitors in both species was similar. 5 Biochemical studies indicated that PDE IV was the major PDE isoform present in the human bronchial tissue. PDEs I, II and V were also identified. 6 Theophylline and pentoxifylline were, as expected, non‐selective inhibitors of the human enzymes, but there was a good correlation between PDE IV inhibitory and bronchorelaxation potencies, suggesting that PDE IV inhibition is important for the clinical bronchodilator activities of the two xanthine compounds. 7 We have confirmed the ability of selective PDE IV inhibitors to cause bronchodilatation in guinea‐pigs in vivo. 8 We conclude that our study has provided further evidence that selective PDE IV inhibitors could act as bronchodilators in the clinic.

Phosphodiesterase 4 inhibition decreases MUC5AC expression induced by epidermal growth factor in human airway epithelial cells

Background: A common pathological feature of chronic inflammatory airway diseases such as asthma and chronic obstructive pulmonary disease (COPD) is mucus hypersecretion. MUC5AC is the predominant mucin gene expressed in healthy airways and is increased in asthmatic and COPD patients. Recent clinical trials indicate that phosphodiesterase type 4 (PDE4) inhibitors may have therapeutic value for COPD and asthma. However, their direct effects on mucin expression have been scarcely investigated. Methods: MUC5AC mRNA and protein expression were examined in cultured human airway epithelial cells (A549) and in human isolated bronchial tissue stimulated with epidermal growth factor (EGF; 25 ng/ml). MUC5AC mRNA was measured by real time RT-PCR and MUC5AC protein by ELISA (cell lysates and tissue homogenates), Western blotting (tissue homogenates) and immunohistochemistry. Results: EGF increased MUC5AC mRNA and protein expression in A549 cells. PDE4 inhibitors produced a concentration dependent inhibition of the EGF induced MUC5AC mRNA and protein expression with potency values (−log IC50): roflumilast (∼7.5) > rolipram (∼6.5) > cilomilast (∼5.5). Roflumilast also inhibited the EGF induced expression of phosphotyrosine proteins, EGF receptor, and phospho-p38- and p44/42-MAPK measured by Western blot analysis in A549 cells. In human isolated bronchus, EGF induced MUC5AC mRNA and protein expression was inhibited by roflumilast (1 μM) as well as the MUC5AC positive staining shown by immunohistochemistry. Conclusion: Selective PDE4 inhibition is effective in decreasing EGF induced MUC5AC expression in human airway epithelial cells. This effect may contribute to the clinical efficacy of this new drug category in mucus hypersecretory diseases.

Characterization of Aclidinium Bromide, a Novel Inhaled Muscarinic Antagonist, with Long Duration of Action and a Favorable Pharmacological Profile

Aclidinium bromide is a novel potent, long-acting inhaled muscarinic antagonist in development for the treatment of chronic obstructive pulmonary disease. Aclidinium showed subnanomolar affinity for the five human muscarinic receptors (M1–M5). [3H]Aclidinium dissociated slightly faster from M2 and M3 receptors than [3H]tiotropium but much more slowly than [3H]ipratropium. Its association rate for the M3 receptor was similar to [3H]ipratropium and 2.6 times faster than [3H]tiotropium. Residence half-life of [3H]aclidinium at the M2 receptor was shorter than at the M3 receptor, demonstrating kinetic selectivity for the M3 receptor. In isolated guinea pig trachea, aclidinium showed comparable potency to ipratropium and tiotropium, faster onset of action than tiotropium, and duration of action similar to tiotropium and significantly longer than ipratropium. Nebulized aclidinium inhibited bronchoconstriction induced by acetylcholine in guinea pigs in a concentration-dependent manner with an onset of action faster than tiotropium. Duration of action of aclidinium (t1/2 = 29 h) was much longer than ipratropium (8 h) but shorter than tiotropium (64 h). In dogs, aclidinium induced a smaller and more transient increase in heart rate than tiotropium at comparable supratherapeutic doses. Therefore, under these conditions, aclidinium showed a greater therapeutic index than tiotropium (4.2 versus 1.6). These results indicate that aclidinium is a potent muscarinic antagonist with a fast onset of action, a long duration of effect, and a favorable cardiovascular safety profile.

In vivo antioxidant treatment protects against bleomycin-induced lung damage in rats

This study examines the activity of the antioxidant N‐acetylcysteine on bleomycin‐induced pulmonary fibrosis in rats with emphasis on the early inflammatory phase. Rats receiving N‐acetylcysteine (300 mg kg−1 day−1, intraperitoneal) had less augmented lung wet weight, and lower levels of proteins, lactate dehydrogenase, neutrophil and macrophage counts in bronchoalveolar lavage fluid and lung myeloperoxidase activity with a betterment of histological score at 3 days postbleomycin. A diminished lung GSH/GSSG ratio and augmented lipid hydroperoxides were observed 3 days postbleomycin. These changes were attenuated by N‐acetylcysteine. Alveolar macrophages from bleomycin‐exposed rats released augmented amounts of superoxide anion and nitric oxide. N‐Acetylcysteine did not modify superoxide anion generation but reduced the increased production of nitric oxide. N‐Acetylcysteine suppressed the bleomycin‐induced increased activation of lung NF‐κB (shift assay and immunohistochemistry), and decreased the augmented levels of the early inflammatory cytokines, tumour necrosis factor‐α, interleukin‐β, interleukin‐6 and macrophage inflammatory protein‐2 observed in bronchoalveolar lavage fluid at 1 and 3 days postbleomycin exposure. At 15 days postbleomycin, N‐acetylcysteine decreased collagen deposition in bleomycin‐exposed rats (hydroxyproline content: 6351±669 and 4626±288 μg per lung in drug vehicle‐ and N‐acetylcysteine‐treated rats, respectively; P<0.05). Semiquantitative histological assessment at this stage showed less collagen deposition in N‐acetylcysteine‐treated rats compared to those receiving bleomycin alone. These results indicate that N‐acetylcysteine reduces the primary inflammatory events, thus preventing cellular damage and the subsequent development of pulmonary fibrosis in the bleomycin rat model.

Mucus and MUC in asthma

Purpose of review Asthma is characterized by chronic airway inflammation and a mucus hypersecretory phenotype comprising excess mucus secretion, goblet cell hyperplasia and submucosal gland hypertrophy. This augmented mucus secretion has been relatively undervalued in asthma compared with airway inflammation. However, mucus plugging contributes to airflow limitation and airway hyperresponsiveness, and to morbidity and mortality in asthma. We review recent contributions to this field and therapeutic avenues to control mucus hypersecretion. Recent findings A distinct mucus hypersecretory phenotype may present in asthma. Overexpression of MUC5AC, MUC5B and MUC2 have been described in asthma secretions, but identification of defined biochemical abnormalities and polymorphisms of mucin genes linked to asthma remains elusive. Activation of epidermal growth factor receptor (EGFR) activation appears central in transducing many different stimuli, including oxidative stress, proteases and cytokines. In contrast, nitrosative stress has barely been investigated. The existence of crosstalk between EGFR and other receptor systems may provide new clues regarding the activity of acetylcholine, adenosine and other agonists of G-protein-coupled receptors and other receptor families on mucin secretion. Modern techniques for noninvasive detection of mucus pathology will advance clinical research in this field. Summary Airway mucus hypersecretion as a part of airway remodelling represents a problem in asthma, and studies of pathophysiology and therapeutic approaches are therefore warranted. Identification of targets such as the EGFR cascade, which are crucial in excessive and abnormal mucus secretion, may lead to the rational design of new antihypersecretory drugs that may enhance future asthma treatment.

Attenuation by oral N-acetylcysteine of bleomycin-induced lung injury in rats

Antioxidant therapy may be useful in diseases with impaired oxidant-antioxidant balance such as pulmonary fibrosis. This study examines the effect of N-acetylcysteine (NAC) on bleomycin-induced lung fibrosis in rats. NAC (3 mmol x kg(-1); oral) was given daily from 1 week prior to a single intratracheal instillation of bleomycin (2.5 U x kg(-1)) or saline, until 14 days postinstillation. NAC partially decreased the augmented collagen deposition in bleomycin-exposed rats (hydroxyproline content was 4,354+/-386 and 3,416+/-326 microg x lung(-1) in vehicle-treated and NAC-treated rats, respectively; p < 0.05). The histological assessment using a semiquantitative score showed less collagen deposition and inflammatory cells in NAC-treated rats compared to those receiving bleomycin alone. NAC failed to inhibit the bleomycin-induced increases in lung wet weight and in cell counts and protein levels of bronchoalveolar lavage fluid, but significantly increased total glutathione and taurine levels in bronchoalveolar lavage fluid. These results indicate that oral N-acetylcysteine improves the pulmonary antioxidant protection and may be useful in reducing lung damage produced by bleomycin.

Angiotensin II Induces Neutrophil Accumulation In Vivo Through Generation and Release of CXC Chemokines

Background—Angiotensin II (Ang II) is implicated in the development of cardiac ischemic disorders in which prominent neutrophil accumulation occurs. Ang II can be generated intravascularly by the renin-angiotensin system or extravascularly by mast cell chymase. In this study, we characterized the ability of Ang II to induce neutrophil accumulation. Methods and Results—Intraperitoneal administration of Ang II (1 nmol/L) induced significant neutrophil recruitment within 4 hours (13.3±2.3×106 neutrophils per rat versus 0.7±0.5×106 in control animals), which disappeared by 24 hours. Maximal levels of CXC chemokines were detected 1 hour after Ang II injection (577±224 pmol/L cytokine-inducible neutrophil chemoattractant [CINC]/keratinocyte-derived chemokine [KC] versus 5±3, and 281±120 pmol/L macrophage inflammatory protein [MIP-2] versus 14±6). Intravital microscopy within the rat mesenteric microcirculation showed that the short-term (30 to 60 minutes) leukocyte–endothelial cell interactions induced by Ang II were attenuated by an anti-rat CINC/KC antibody and nearly abolished by the CXCR2 antagonist SB-517785-M. In human umbilical vein endothelial cells (HUVECs) or human pulmonary artery media in culture, Ang II induced interleukin (IL)-8 mRNA expression at 1, 4, and 24 hours and the release of IL-8 at 4 hours through interaction with Ang II type 1 receptors. When HUVECs were pretreated with IL-1 for 24 hours to promote IL-8 storage in Weibel-Palade bodies, the Ang II–induced IL-8 release was more rapid and of greater magnitude. Conclusions—Ang II provokes rapid neutrophil recruitment, mediated through the release of CXC chemokines such as CINC/KC and MIP-2 in rats and IL-8 in humans, and may contribute to the infiltration of neutrophils observed in acute myocardial infarction.

Menopause and Ovariectomy Cause a Low Grade of Systemic Inflammation that May Be Prevented by Chronic Treatment with Low Doses of Estrogen or Losartan

The incidence of cardiovascular diseases in premenopausal women is lower than in men or postmenopausal women. This study reports the discovery of a low grade of systemic inflammation, including monocyte adhesion to arterial endothelium, elicited by menopause or estrogen depletion. Chronic treatment with low dose of 17-β-estradiol or inhibition of the renin-angiotensin system reduced this inflammation. Using an in vitro flow chamber system with human arterial and venous endothelial cells, we found that leukocytes from healthy postmenopausal women were more adhesive to the arterial endothelium than those from premenopausal women regardless of the stimulus used on endothelial cells. Increased circulating levels of IL-8, MCP-1, RANTES, and MIP-1α and monocyte CD11b expression were also encountered in postmenopausal vs premenopausal subjects. This translational data led us to investigate the mechanisms in Sprague-Dawley rats. Using intravital microscopy, we imaged mesenteric arterioles and found significant increases in arteriolar leukocyte adhesion, cell adhesion molecule expression, and plasma levels of cytokine-induced neutrophil chemoattractant (CINC/KC), MCP-1, and MIP-1α in 1-mo ovariectomized rats. Chronic treatment of ovariectomized rats with low dose of 17-β-estradiol, losartan, both, or benazepril inhibited ovariectomy-induced arteriolar mononuclear leukocyte adhesion by 77%, 58%, 92%, and 65% respectively, partly by inhibition of cell adhesion molecule up-regulation and the increase in circulating chemokines. These results demonstrate that menopause and ovariectomy generate a low grade of systemic inflammation. Therefore, administration of low doses of estrogens or inhibition of the renin-angiotensin system, at early stages of estrogen deficiency, might prevent the systemic inflammation associated with menopause and decrease the risk of suffering further cardiovascular diseases.

Angiotensin II-Induced Mononuclear Leukocyte Interactions with Arteriolar and Venular Endothelium Are Mediated by the Release of Different CC Chemokines

Angiotensin II (Ang-II) is associated with atherogenesis and arterial subendothelial mononuclear leukocyte infiltration. We have demonstrated that Ang-II causes the initial attachment of mononuclear cells to the arteriolar endothelium. We now report on the contribution of CC chemokines to this response. Intraperitoneal administration of 1 nM Ang-II induced MCP-1, RANTES, and MIP-1α generation, maximal at 4 h, followed by mononuclear leukocyte recruitment at 8 and 24 h. Using intravital microscopy within the rat mesenteric microcirculation 4 h after exposure to 1 nM Ang-II, arteriolar mononuclear cell adhesion was 80–90% inhibited by pretreatment with Met-RANTES, a CCR1 and CCR5 antagonist, or an anti-MCP-1 antiserum, without affecting the increased endothelial expression of P-selectin and VCAM-1. Conversely, leukocyte interactions with the venular endothelium, although inhibited by Met-RANTES, were little affected by the anti-MCP-1. Using rat whole blood in vitro, Ang-II (100 nM) induced the expression of monocyte CD11b that was inhibited by Met-RANTES but not by anti-MCP-1. Stimulation of human endothelial cells (human umbilical arterial endothelial cells and HUVECs) with 1–1000 nM Ang-II, predominantly acting at its AT1 receptor, induced the release of MCP-1 within 1 h, RANTES within 4 h, and MCP-3 within 24 h. Eotaxin-3, a natural CCR2 antagonist, was released within 1 h and may delay mononuclear cell responses to MCP-1. Therefore, Ang-II-induced mononuclear leukocyte recruitment at arterioles and venules is mediated by the production of different CC chemokines. Thus, Ang-II may be a key molecule in the initial attachment of mononuclear cells to the arterial endothelium in cardiovascular disease states where this event is a characteristic feature.

Oral N‐acetylcysteine reduces bleomycin-induced lung damage and mucin Muc5ac expression in rats

Oxidative stress is involved in the pathogenesis of pulmonary fibrosis, therefore antioxidants may be of therapeutic value. Clinical work indicates that N‐acetylcysteine (NAC) may be beneficial in this disease. The activity of this antioxidant was examined on bleomycin-induced lung damage, mucus secretory cells hyperplasia and mucin Muc5ac gene expression in rats. NAC (3 mmol·kg−1·day−1) or saline was given orally to Sprague-Dawley rats for 1 week prior to a single intratracheal instillation of bleomycin (2.5 U·kg−1) and for 14 days postinstillation. NAC decreased collagen deposition in bleomycin-exposed rats (hydroxyproline content was 4,257±323 and 3,200±192 µg·lung−1 in vehicle- and NAC‐treated rats, respectively) and lessened the fibrotic area assessed by morphometric analysis. The bleomycin-induced increases in lung tumour necrosis factor‐α and myeloperoxidase activity were reduced by NAC treatment. The numbers of mucus secretory cells in airway epithelium, and the Muc5ac messenger ribonucleic acid and protein expression, were markedly augmented in rats exposed to bleomycin. These changes were significantly reduced in NAC‐treated rats. These results indicate that bleomycin increases the number of airway secretory cells and their mucin production, and that oral N‐acetylcysteine improved pulmonary lesions and reduced the mucus hypersecretion in the bleomycin rat model.