Delivery system for budesonide based on lipid-DNA.

Budesonide is a hydrophobic glucocorticoid with high anti-inflammatory activity for the treatment of asthma, inflammatory bowel disease and rheumatoid arthritis. A micellar drug-delivery system based on lipid-DNA may provide a strategy to maximize its drug efficacy and reduce adverse effects. In this work, we report the use of lipid-DNAA (UU11mer), featuring two hydrophobic alkyl chains and forming micelles at a comparatively low critical micelle concentration, to render budesonide water-soluble with a high loading capacity (LC). The inhibition of interleukin-8 (IL-8) release shows that the new delivery system retains the inhibitory activity in cell-based assays. In conclusion, this research provides a novel approach to formulate and administer budesonide in a non-invasive manner, which dramatically improves its water-solubility while retaining its bioavailability.

The PDE4 inhibitor CHF-6001 and LAMAs inhibit bronchoconstriction-induced remodeling in lung slices.

Combination therapy of PDE4 inhibitors and anticholinergics induces bronchoprotection in COPD. Mechanical forces that arise during bronchoconstriction may contribute to airway remodeling. Therefore, we investigated the impact of PDE4 inhibitors and anticholinergics on bronchoconstriction-induced remodeling. Because of the different mechanism of action of PDE4 inhibitors and anticholinergics, we hypothesized functional interactions of these two drug classes. Guinea pig precision-cut lung slices were preincubated with the PDE4 inhibitors CHF-6001 or roflumilast and/or the anticholinergics tiotropium or glycopyorrolate, followed by stimulation with methacholine (10 µM) or TGF-ß1 (2 ng/ml) for 48 h. The inhibitory effects on airway smooth muscle remodeling, airway contraction, and TGF-ß release were investigated. Methacholine-induced protein expression of smooth muscle-myosin was fully inhibited by CHF-6001 (0.3-100 nM), whereas roflumilast (1 µM) had smaller effects. Tiotropium and glycopyrrolate fully inhibited methacholine-induced airway remodeling (0.1-30 nM). The combination of CHF-6001 and tiotropium or glycopyrrolate, in concentrations partially effective by themselves, fully inhibited methacholine-induced remodeling in combination. CHF-6001 did not affect airway closure and had limited effects on TGF-ß1-induced remodeling, but rather, it inhibited methacholine-induced TGF-ß release. The PDE4 inhibitor CHF-6001, and to a lesser extent roflumilast, and the LAMAs tiotropium and glycopyrrolate inhibit bronchoconstriction-induced remodeling. The combination of CHF-6001 and anticholinergics was more effective than the individual compounds. This cooperativity might be explained by the distinct mechanisms of action inhibiting TGF-ß release and bronchoconstriction.

Bronchoconstriction Induces TGF-ß Release and Airway Remodelling in Guinea Pig Lung Slices.

Airway remodelling, including smooth muscle remodelling, is a primary cause of airflow limitation in asthma. Recent evidence links bronchoconstriction to airway remodelling in asthma. The mechanisms involved are poorly understood. A possible player is the multifunctional cytokine TGF-ß, which plays an important role in airway remodelling. Guinea pig lung slices were used as an in vitro model to investigate mechanisms involved in bronchoconstriction-induced airway remodelling. To address this aim, mechanical effects of bronchoconstricting stimuli on contractile protein expression and TGF-ß release were investigated. Lung slices were viable for at least 48 h. Both methacholine and TGF-ß1 augmented the expression of contractile proteins (sm-α-actin, sm-myosin, calponin) after 48 h. Confocal fluorescence microscopy showed that increased sm-myosin expression was enhanced in the peripheral airways and the central airways. Mechanistic studies demonstrated that methacholine-induced bronchoconstriction mediated the release of biologically active TGF-ß, which caused the increased contractile protein expression, as inhibition of actin polymerization (latrunculin A) or TGF-ß receptor kinase (SB431542) prevented the methacholine effects, whereas other bronchoconstricting agents (histamine and KCl) mimicked the effects of methacholine. Collectively, bronchoconstriction promotes the release of TGF-ß, which induces airway smooth muscle remodelling. This study shows that lung slices are a useful in vitro model to study mechanisms involved in airway remodelling.

A new perspective on muscarinic receptor antagonism in obstructive airways diseases.

Acetylcholine has traditionally only been regarded as a neurotransmitter of the parasympathetic nervous system, causing bronchoconstriction and mucus secretion in asthma and COPD by muscarinic receptor activation on airway smooth muscle and mucus-producing cells. Recent studies in experimental models indicate that muscarinic receptor stimulation in the airways also induces pro-inflammatory, pro-proliferative and pro-fibrotic effects, which may involve activation of airway structural and inflammatory cells by neuronal as well as non-neuronal acetylcholine. In addition, mechanical changes caused by muscarinic agonist-induced bronchoconstriction may be involved in airway remodeling. Crosstalk between muscarinic receptors and ß2-adrenoceptors on airway smooth muscle causes a reduced bronchodilator response to ß2-agonists, and a similar mechanism could possibly apply to the poor inhibition of inflammatory and remodeling processes by these drugs. Collectively, these findings provide novel perspectives for muscarinic receptor antagonists in asthma and COPD, since these drugs may not only acutely affect cholinergic airways obstruction, but also have important beneficial effects on ß2-agonist responsiveness, airway inflammation and remodeling. The clinical relevance of these findings is presently under investigation and starting to emerge.

Cross-talk between transforming growth factor-ß1 and muscarinic M2 receptors augments airway smooth muscle proliferation.

Transforming growth factor-ß1 (TGF-ß1) is a central mediator in tissue remodeling processes, including fibrosis and airway smooth muscle (ASM) hyperplasia, as observed in asthma. The mechanisms underlying this response, however, remain unclear because TGF-ß1 exerts only weak mitogenic effects on ASM cells. In this study, we hypothesized that the mitogenic effect of TGF-ß1 on ASM is indirect and requires prolonged exposure to allow for extracellular matrix (ECM) deposition. To address this hypothesis, we investigated the effects of acute and prolonged treatment with TGF-ß1, alone and in combination with the muscarinic receptor agonist methacholine, on human ASM cell proliferation. Acutely, TGF-ß1 exerted no mitogenic effect. However, prolonged treatment (for 7 d) with TGF-ß1 increased ASM cell proliferation and potentiated the platelet-derived growth factor-induced mitogenic response. Muscarinic receptor stimulation with methacholine synergistically enhanced the effect of TGF-ß1. Interestingly, the integrin-blocking peptide Arg-Gly-Asp-Ser, as well as integrin α5ß1 function-blocking antibodies, inhibited the effects of TGF-ß1 and its combination with methacholine on cell proliferation. Accordingly, prolonged treatment with TGF-ß1 increased fibronectin expression, which was also synergistically enhanced by methacholine. The synergistic effects of methacholine on TGF-ß1-induced proliferation were reduced by the long-acting muscarinic receptor antagonist tiotropium and the M2 receptor subtype-selective antagonist gallamine, but not the M3-selective antagonist DAU5884. In line with these findings, the irreversible Gi protein inhibitor pertussis toxin also prevented the potentiation of TGF-ß1-induced proliferation by methacholine. We conclude that prolonged exposure to TGF-ß1 enhances ASM cell proliferation, which is mediated by extracellular matrix-integrin interactions, and which can be enhanced by muscarinic M2 receptor stimulation.

Muscarinic receptor stimulation augments TGF-ß1-induced contractile protein expression by airway smooth muscle cells.

Acetylcholine (ACh) is the primary parasympathetic neurotransmitter in the airways. Recently, it was established that ACh, via muscarinic receptors, regulates airway remodeling in animal models of asthma and chronic obstructive pulmonary disease (COPD). The mechanisms involved are not well understood. Here, we investigated the functional interaction between muscarinic receptor stimulation and transforming growth factor (TGF)-ß(1) on the expression of contractile proteins in human airway smooth muscle (ASM) cells. ASM cells expressing functional muscarinic M(2) and M(3) receptors were stimulated with methacholine (MCh), TGF-ß(1), or their combination for up to 7 days. Western blot analysis revealed a strong induction of sm-α-actin and calponin by TGF-ß(1), which was increased by MCh in ASM cells. Immunocytochemistry confirmed these results and revealed that the presence of MCh augmented the formation of sm-α-actin stress fibers by TGF-ß(1). MCh did not augment TGF-ß(1)-induced gene transcription of contractile phenotype markers. Rather, translational processes were involved in the augmentation of TGF-ß(1)-induced contractile protein expression by muscarinic receptor stimulation, including phosphorylation of glycogen synthase kinase-3ß and 4E-binding protein 1, which was enhanced by MCh. In conclusion, muscarinic receptor stimulation augments functional effects of TGF-ß(1) in human ASM cells on cellular processes that underpin ASM remodeling in asthma and COPD.

Regulation of airway inflammation and remodeling by muscarinic receptors: perspectives on anticholinergic therapy in asthma and COPD.

Acetylcholine is the primary parasympathetic neurotransmitter in the airways and an autocrine/paracrine secreted hormone from non-neuronal origins including inflammatory cells and airway structural cells. In addition to the well-known functions of acetylcholine in regulating bronchoconstriction and mucus secretion, it is increasingly evident that acetylcholine regulates inflammatory cell chemotaxis and activation, and also participates in signaling events leading to chronic airway wall remodeling that is associated with chronic obstructive airway diseases including asthma and COPD. As muscarinic receptors appear responsible for most of the pro-inflammatory and remodeling effects of acetylcholine, these findings have significant implications for anticholinergic therapy in asthma and COPD, which is selective for muscarinic receptors. Here, the regulatory role of acetylcholine in inflammation and remodeling in asthma and COPD will be discussed including the perspectives that these findings offer for anticholinergic therapy in these diseases.

Inhibition of IGF-1R-dependent PI3K activation sensitizes colon cancer cells specifically to DR5-mediated apoptosis but not to rhTRAIL.

BACKGROUND: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) initiates apoptosis in tumor cells upon binding to its cognate agonistic receptors, death receptors 4 and 5 (DR4 and DR5). The activity of the insulin-like growth factor 1 (IGF-1) survival pathway is often increased in cancer, influencing both cell proliferation and apoptosis. We hypothesized that inhibiting the IGF-1 receptor (IGF-1R) using NVP-AEW541, a small molecular weight tyrosine kinase inhibitor of the IGF-1R, could increase death receptor (DR)-mediated apoptosis in colon cancer cells. METHODS: The analyses were performed by caspase assay, flow cytometry, Western blotting, immunoprecipitation and fluorescent microscopy. RESULTS: Preincubation with NVP-AEW541 surprisingly decreased apoptosis induced by recombinant human TRAIL (rhTRAIL) or an agonistic DR4 antibody while sensitivity to an agonistic DR5 antibody was increased. NVP-AEW541 could inhibit IGF-1-induced activation of the phosphatidylinositol 3-kinase (PI3K) pathway. The effects of the PI3K inhibitor LY294002 on TRAIL-induced apoptosis were similar to those of NVP-AEW541, further supporting a role for IGF-1R-mediated activation of PI3K. We show that PI3K inhibition enhances DR5-mediated caspase 8 processing but also lowers DR4 membrane expression and DR4-mediated caspase 8 processing. Inhibition of PI3K reduced rhTRAIL sensitivity independently of the cell line preference for either DR4- or DR5-mediated apoptosis signaling. CONCLUSIONS: Our study indicates that individual effects on DR4 and DR5 apoptosis signaling should be taken into consideration when combining DR-ligands with PI3K inhibition.

Inhibition of IGF-1R-dependent PI3K activation sensitizes colon cancer cells specifically to DR5-mediated apoptosis but not to rhTRAIL.

BACKGROUND: tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) initiates apoptosis in tumor cells upon binding to its cognate agonistic receptors, death receptors 4 and 5 (DR4 and DR5). The activity of the insulin-like growth factor 1 (IGF-1) survival pathway is often increased in cancer, influencing both cell proliferation and apoptosis. We hypothesized that inhibiting the IGF-1 receptor (IGF-1R) using NVP-AEW541, a small molecular weight tyrosine kinase inhibitor of the IGF-1R, could increase death receptor (DR)-mediated apoptosis in colon cancer cells. METHODS: the analyses were performed by caspase assay, flow cytometry, Western blotting, immunoprecipitation and fluorescent microscopy. RESULTS: preincubation with NVP-AEW541 surprisingly decreased apoptosis induced by recombinant human TRAIL (rhTRAIL) or an agonistic DR4 antibody while sensitivity to an agonistic DR5 antibody was increased. NVP-AEW541 could inhibit IGF-1-induced activation of the phosphatidylinositol 3-kinase (PI3K) pathway. The effects of the PI3K inhibitor LY294002 on TRAIL-induced apoptosis were similar to those of NVP-AEW541, further supporting a role for IGF-1R-mediated activation of PI3K. We show that PI3K inhibition enhances DR5-mediated caspase 8 processing but also lowers DR4 membrane expression and DR4-mediated caspase 8 processing. Inhibition of PI3K reduced rhTRAIL sensitivity independently of the cell line preference for either DR4- or DR5-mediated apoptosis signaling. CONCLUSIONS: our study indicates that individual effects on DR4 and DR5 apoptosis signaling should be taken into consideration when combining DR-ligands with PI3K inhibition.

Pro-inflammatory mechanisms of muscarinic receptor stimulation in airway smooth muscle.

BACKGROUND: Acetylcholine, the primary parasympathetic neurotransmitter in the airways, plays an important role in bronchoconstriction and mucus production. Recently, it has been shown that acetylcholine, by acting on muscarinic receptors, is also involved in airway inflammation and remodelling. The mechanism(s) by which muscarinic receptors regulate inflammatory responses are, however, still unknown. METHODS: The present study was aimed at characterizing the effect of muscarinic receptor stimulation on cytokine secretion by human airway smooth muscle cells (hASMc) and to dissect the intracellular signalling mechanisms involved. hASMc expressing functional muscarinic M2 and M3 receptors were stimulated with the muscarinic receptor agonist methacholine, alone, and in combination with cigarette smoke extract (CSE), TNF-α, PDGF-AB or IL-1ß. RESULTS: Muscarinic receptor stimulation induced modest IL-8 secretion by itself, yet augmented IL-8 secretion in combination with CSE, TNF-α or PDGF-AB, but not with IL-1ß. Pretreatment with GF109203X, a protein kinase C (PKC) inhibitor, completely normalized the effect of methacholine on CSE-induced IL-8 secretion, whereas PMA, a PKC activator, mimicked the effects of methacholine, inducing IL-8 secretion and augmenting the effects of CSE. Similar inhibition was observed using inhibitors of IκB-kinase-2 (SC514) and MEK1/2 (U0126), both downstream effectors of PKC. Accordingly, western blot analysis revealed that methacholine augmented the degradation of IκBα and the phosphorylation of ERK1/2 in combination with CSE, but not with IL-1ß in hASMc. CONCLUSIONS: We conclude that muscarinic receptors facilitate CSE-induced IL-8 secretion by hASMc via PKC dependent activation of IκBα and ERK1/2. This mechanism could be of importance for COPD patients using anticholinergics.

Public TCR use by herpes simplex virus-2-specific human CD8 CTLs.

Recombination of germline TCR alpha and beta genes generates polypeptide receptors for MHC peptide. Ag exposure during long-term herpes simplex infections may shape the T cell repertoire over time. We investigated the CD8 T cell response to HSV-2 in chronically infected individuals by sequencing the hypervariable regions encoding TCR alpha and beta polypeptides from T cell clones recognizing virion protein 22 aa 49-57, an immunodominant epitope. The most commonly detected TCRBV gene segment, found in four of five subjects and in 12 of 50 independently derived T cell clones, was TCRBV12-4. Nineteen to seventy-two percent of tetramer-binding cells in PBMCs were stained ex vivo with a TCRBV12 mAb. Three alpha-chain and three beta-chain public TCR sequences were shared between individuals. Public heterodimers were also detected. Promiscuous pairing of a specific TCRVA1-1 sequence with several different TCRB polypeptides was observed, implying a dominant structural role for the TCRA chain for these clonotypes. Functional avidity for cytotoxicity and IFN-gamma release was relatively invariant, except for one subject with both high avidity and unique TCR sequences and lower HSV-2 shedding. These data indicate that the CD8 response to a dominant alpha-herpesvirus epitope converges on preferred TCR sequences with relatively constant functional avidity.

De novo synthesis of {beta}-catenin via H-Ras and MEK regulates airway smooth muscle growth.

beta-Catenin is a component of adherens junctions that also acts as a transcriptional coactivator when expressed in the nucleus. Growth factors are believed to regulate the nuclear expression of beta-catenin via inactivation of glycogen synthase kinase 3 (GSK-3) by phosphorylation, resulting in increased beta-catenin protein stability. Here, we report on a novel pathway that regulates the expression and nuclear presence of beta-catenin. In proliferating human airway smooth muscle cells, we observed increased expression of beta-catenin, which was required for proliferation. Interestingly, increased beta-catenin expression was accompanied by an increase in beta-catenin mRNA and was independent of beta-catenin liberation from the plasma membrane, suggesting a role for de novo synthesis. This was confirmed using actinomycin D and cycloheximide, which abrogated the induction and nuclear localization of beta-catenin protein. GSK-3 inhibition using SB216763 failed to regulate beta-catenin mRNA. However, expression of dominant negative H-Ras or pharmacological inhibition of MEK reduced serum and TGF-beta-induced beta-catenin mRNA and protein. Collectively, these data indicate that beta-catenin is an important signaling intermediate in airway smooth muscle growth and that its cellular accumulation and nuclear localization require de novo protein synthesis effected, in part, via H-Ras and MEK.-Gosens, R., Baarsma, H. A., Heijink, I. H., Oenema, T. A., Halayko, A. J., Meurs, H., Schmidt, M. De novo synthesis of beta-catenin via H-Ras and MEK regulates airway smooth muscle growth.