Repairing damaged tissues is an essential homeostatic mechanism that enables clearance of dead or damaged cells after injury, and the maintenance of tissue integrity. However, exaggeration of this process in the lung can lead to the development of fibrotic scar tissue. This is characterized by excessive accumulation of extracellular matrix (ECM) components such as fibronectin, proteoglycans, hyaluronic acid, and interstitial collagens. After tissue injury, or a breakdown of tissue integrity, a cascade of events unfolds to maintain normal tissue homeostasis. Inflammatory mediators are released from injured epithelium, leading to both platelet activation and inflammatory cell migration. Inflammatory cells are capable of releasing multiple pro-inflammatory and fibrogenic mediators such as transforming growth factor (TGF)β and interleukin (IL)-13, which can trigger myofibroblast proliferation and recruitment. The myofibroblast population is also expanded as a result of epithelial cells undergoing epithelial-to-mesenchymal transition and of the activation of resident fibroblasts, leading to ECM deposition and tissue remodeling. In the healthy lung, wound healing then proceeds to restore the normal architecture of the lung; however, fibrosis can develop when the wound is severe, the tissue injury persists, or the repair process becomes dysregulated. Understanding the processes regulating aberrant wound healing and the matrix in the chronic fibrotic lung disease idiopathic pulmonary fibrosis (IPF), is key to identifying new treatments for this chronic debilitating disease. This review focuses primarily on the emerging role of enzymes in the lungs of patients with IPF. Elevated expression of a number of enzymes that can directly modulate the ECM has been reported, and recent data indicates that modulating the activity of these enzymes can have a downstream effect on fibrotic tissue remodeling.

The cardiovascular effects of N6-2-(4-aminophenyl)ethyladenosine (APNEA), which when radiolabelled with 1251 shows high affinity for the newly described adenosine A3 receptor, have been investigated in the angiotension IT-supported circulation of the pithed rat. APNEA induces hypotensive responses which are unaffected by high doses (20-40 mg kg-') of the broad spectrum, adenosine receptor antagonist, 8-(p-sulphophenyl)theophylline (8-SPT). 8-SPT-resistant falls in blood pressure are also seen, in the absence of bradycardia, with 5'-N-ethylcarboxamidoadenosine (NECA) and the R-and S-enantiomers of N6-phenylisopropyladenosine (PIA). Xanthine insensitivity, high potencies of APNEA, NECA and R-PIA, and an enantiomeric selectivity favouring R-over S-PIA are distinguishing features of the adenosine A3 receptor. We suggest that hypotension in the pithed rat may be a functional correlate of this site.

Calcitonin gene-related peptide (CGRP), a potent vasodilator, has been implicated in the pathogenesis of migraine. Its release from adult rat trigeminal neurons in culture was shown to be markedly increased by the activation of adenylate cyclase with forskolin. Modulation of this secretion was investigated by a number of agents with known inhibitory effects on cAMP generation mediated via receptor coupling to G(i/o) proteins. Significantly, forskolin-stimulated CGRP release could be closely correlated with the phosphorylation of the protein kinase A (PKA) substrate cyclic AMP response element-binding protein (CREB). Forskolin-stimulated CGRP release could be potently and effectively inhibited by the adenosine A(1) receptor-selective agonist GR79236X (pIC(50) = 7.7 +/- 0.1, maximal inhibition 65 +/- 2.5% at 300 nM), whereas the A(2A) (CGS21680) and the A(3) (2-chloro-N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide) receptor-selective agonists were without effect. GR79236X-mediated inhibition was abolished by the A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine. Immunocytochemical studies and Western analysis revealed the presence of adenosine A(1) receptors on trigeminal neurons. However, despite the additional detection of 5-hydroxytryptamine (5-HT)(1B) receptors on these cells, the clinically effective antimigraine 5-HT(1B/1D) agonist sumatriptan did not inhibit forskolin-stimulated CGRP release nor did it show any effect on the concomitant CREB phosphorylation. In contrast, the mu-opioid agonist fentanyl elicited a 74 +/- 4% reduction in CGRP levels. Forskolin-stimulated CGRP release and CREB phosphorylation could be mimicked by incubation of the cells with chlorophenylthio-cAMP and blocked by pretreatment with the PKA inhibitor myrPKI(14-22). Taken together, the present data confirm the PKA-dependence of forskolin-stimulated CGRP release and suggest that A(1) adenosine agonists may warrant further investigation in models of migraine and neurogenic inflammation.

Human rhinoviruses (HRV) cause the majority of common colds and acute exacerbations of asthma and chronic obstructive pulmonary disease (COPD). Effective therapies are urgently needed, but no licensed treatments or vaccines currently exist. Of the 100 identified serotypes, ∼90% bind domain 1 of human intercellular adhesion molecule-1 (ICAM-1) as their cellular receptor, making this an attractive target for development of therapies; however, ICAM-1 domain 1 is also required for host defence and regulation of cell trafficking, principally via its major ligand LFA-1. Using a mouse anti-human ICAM-1 antibody (14C11) that specifically binds domain 1 of human ICAM-1, we show that 14C11 administered topically or systemically prevented entry of two major groups of rhinoviruses, HRV16 and HRV14, and reduced cellular inflammation, pro-inflammatory cytokine induction and virus load in vivo. 14C11 also reduced cellular inflammation and Th2 cytokine/chemokine production in a model of major group HRV-induced asthma exacerbation. Interestingly, 14C11 did not prevent cell adhesion via human ICAM-1/LFA-1 interactions in vitro, suggesting the epitope targeted by 14C11 was specific for viral entry. Thus a human ICAM-1 domain-1-specific antibody can prevent major group HRV entry and induction of airway inflammation in vivo.

The opposing effects on proliferation mediated by G-protein-coupled receptor isoforms differing in their COOH termini could be correlated with the abilities of the receptors to differentially activate p38, implicated in apoptotic events, or phosphatidylinositol 3-kinase (PI 3-K), which provides a source of survival signals. These contrasting growth responses of the somatostatin sst 2 receptor isoforms, which couple to identical G␣ subunit pools (G␣ i3 > G␣ i2 >> G␣ 0 ), were both inhibited following ␤␥ sequestration. The sst 2(a) receptormediated ATF-2 activation and inhibition of proliferation induced by basic fibroblast growth factor (bFGF) were dependent on prolonged phosphorylation of p38. In contrast, cell proliferation and the associated transient phosphorylation of Akt and p70 rsk induced by sst 2(b) receptors were blocked by the PI 3-K inhibitor LY 294002. Stimulation with bFGF alone had no effect on the activity of either p38 or Akt but markedly enhanced p38 phosphorylation mediated by sst 2(a) receptors, suggesting that a complex interplay exists between the transduction cascades activated by these distinct receptor types. In addition, although all receptors mediated a sustained activation of extracellular signal-regulated kinases (ERK1 and ERK2), induction of the tumor suppressor p21 cip1 was detected only following amplification of ERK and p38 phosphorylation by concomitant bFGF and sst 2(a) receptor activation. Expression of constitutively active Akt in the presence of a p38 inhibitor enabled a proliferative response to be detected in sst 2(a) receptor-expressing cells. These findings demonstrate that the duration of activation and a critical balance between the mitogen-activated protein kinase and PI 3-K pathways are important for controlling cell proliferation and that the COOH termini of the sst 2 receptor isoforms may determine the selection of appropriate ␤␥-pairings necessary for interaction with distinct kinase cascades.Mitogen-activated protein (MAP) kinases are proline-directed serine/threonine kinases that play important roles as mediators of cellular responses to a variety of stimuli such as growth factors, cytokines, hormones, and environmental stresses (18,23). MAP kinases in mammalian cells have been classified into at least four subfamilies: extracellular signalregulated kinases (ERKs), stress-activated protein kinases/cJun NH 2 -terminal kinase (SAPKs/JNK), p38 kinases, and BMK1/ERK5 (51). ERK is activated by many growth factors and cytokines and is implicated in cell growth as well as differentiation (32). Various stressors such as chemical agents and UV irradiation, tumor necrosis factor, interleukin-1, CD40 ligand, and Fas/CD95 ligand stimulate the activities of SAPKs and p38 (10, 24) which appear to play a decisive role in the control of cell death. Thus, the SAPK pathway is critical during ceramide-induced (49) and stress-induced (56) apoptosis as well as in the Daxx-mediated Fas cascade (55), whereas transfection of a constitutively active mutant of MKK3/6, the physiological ac...

Conflict of interest: DKF and LAM are employees of an AstraZeneca group company and may receive AstraZeneca shares as part of their usual remuneration.

1 The mouse somatostatin (SRIF) sst 2 receptor exists in two splice variants, sst 2(a) and sst 2(b) , which di er in their intracellular carboxy-termini only. The murine sst 2(b) receptor was reported to be less prone to agonist-induced desensitization as compared with the sst 2(a) receptor. To determine whether a sst 2(b) splice variant with similar functional characteristics exists in the rat, we have isolated a cDNA fragment from rat gastric mucosa encoding a sst 2(b) receptor and expressed the full-length protein in CHO-K1 cells for functional characterization. 2 This study provides the ®rst evidence for the occurrence in the rat of the sst 2(b) receptor, which has a 15 amino acid carboxy-terminus di ering in composition to the 38 amino acid C-terminus of the rat sst 2(a) receptor. 3 In CHO-K1 cells expressing rat recombinant sst 2(a) or sst 2(b) receptors, SRIF caused concentrationdependent increases in extracellular acidi®cation rates (EAR) with pEC 50 values of 9.0 and 9.9, respectively. Pre-treatment with pertussis toxin (Ptx) caused a rightward displacement of the SRIF concentration-e ect curves with pEC 50 values of 8.3 (sst 2(a) ) and 8.4 (sst 2(b) ). 4 SRIF (3 pM ± 3 nM) also caused concentration-dependent inhibition of forskolin-stimulated cyclic AMP formation in CHO-sst 2(a) cells (pIC 50 10.5) and CHO-sst 2(b) cells (pIC 50 10.4). The degree of inhibition was less with higher concentrations of SRIF resulting in bell-shaped concentration-e ect curves. Following pre-treatment with Ptx, the inhibitory e ect of SRIF was abolished and SRIF caused only increases in cyclic AMP formation. 5 Both the SRIF-induced increases in EAR and inhibition of cyclic AMP formation were susceptible to agonist-induced desensitization, but this was less apparent following pre-treatment with Ptx. 6 This demonstrates that the operational characteristics of the recombinant rat sst 2(a) and sst 2(b) receptors are broadly similar. Both isoforms couple to Ptx-sensitive as well as -insensitive G proteins and are equally prone to agonist-induced desensitization.

BackgroundIdiopathic pulmonary fibrosis (IPF) is a chronic and progressive lung disease which presents a grave prognosis for diagnosed patients. Nintedanib (a triple tyrosine kinase inhibitor) and pirfenidone (unclear mechanism of action) are the only approved therapies for IPF, but have limited efficacy. The pathogenic mechanisms of this disease are not fully elucidated; however, a role for mast cells (MCs) has been postulated.ObjectivesThe aim of this work was to investigate a role for MCs in IPF and to understand whether nintedanib or pirfenidone could impact MC function.Methods and resultsMCs were significantly elevated in human IPF lung and negatively correlated with baseline lung function (FVC). Importantly, MCs were positively associated with the number of fibroblast foci, which has been linked to increased mortality. Furthermore, MCs were increased in the region immediately surrounding the fibroblast foci, and co-culture studies confirmed a role for MC–fibroblast crosstalk in fibrosis. Nintedanib but not pirfenidone inhibited recombinant stem cell factor (SCF)–induced MC survival. Further evaluation of nintedanib determined that it also inhibited human fibroblast-mediated MC survival. This was likely via a direct effect on ckit (SCF receptor) since nintedanib blocked SCF-stimulated ckit phosphorylation, as well as downstream effects on MC proliferation and cytokine release. In addition, nintedanib ablated the increase in lung MCs and impacted high tissue density frequency (HDFm) in a rat bleomycin model of lung fibrosis.ConclusionNintedanib inhibits MC survival and activation and thus provides a novel additional mechanism by which this drug may exert anti-fibrotic effects in patients with IPF.