Epithelial-Mesenchymal Transition Mechanisms in Chronic Airway Diseases: A Common Process to Target?

Epithelial-to-mesenchymal transition (EMT) is a reversible process, in which epithelial cells lose their epithelial traits and acquire a mesenchymal phenotype. This transformation has been described in different lung diseases, such as lung cancer, interstitial lung diseases, asthma, chronic obstructive pulmonary disease and other muco-obstructive lung diseases, such as cystic fibrosis and non-cystic fibrosis bronchiectasis. The exaggerated chronic inflammation typical of these pulmonary diseases can induce molecular reprogramming with subsequent self-sustaining aberrant and excessive profibrotic tissue repair. Over time this process leads to structural changes with progressive organ dysfunction and lung function impairment. Although having common signalling pathways, specific triggers and regulation mechanisms might be present in each disease. This review aims to describe the various mechanisms associated with fibrotic changes and airway remodelling involved in chronic airway diseases. Having better knowledge of the mechanisms underlying the EMT process may help us to identify specific targets and thus lead to the development of novel therapeutic strategies to prevent or limit the onset of irreversible structural changes.

Needle-free iontophoresis-driven ß-adrenergic sweat rate test.

OBJECTIVES: Two CFTR-dependent ß-adrenergic sweat rate tests applying intradermal drug injections were reported to better define diagnosis and efficacy of CFTR-directed therapies. The aim of this work was to develop and test a needle-free image-based test and to provide an accurate analysis of the responses. METHODS: The modified method was conducted by applying two successive iontophoresis sessions using the Macroduct device. Efficiency of drug delivery was tested by evaporimetry. Cholinergically stimulated sweating was evoked by pilocarpine iontophoresis. ß-adrenergically stimulated sweating was obtained by iontophoresis of isoproterenol and aminophylline in the presence of atropine and ascorbic acid. A nonlinear mixed-effects (NLME) approach was applied to model volumes of sweat and subject-specific effects displaying inter- and intra-subject variability. RESULTS: Iontophoresis provided successful transdermal delivery of all drugs, including almost neutral isoproterenol and aminophylline. Pilocarpine was used at a concentration ∼130-times lower than that used in the classical Gibson and Cooke sweat test. Addition of ascorbic acid lowered the pH of the solution, made it stable, prevented isoproterenol degradation and promoted drug iontophoresis. Maximal secretory capacity and kinetic rate of ß-adrenergic responses were blunted in CF. A cutoff of 5.2 minutes for ET50, the time to reach the half maximal secretion, discriminated CF from controls with a 100% sensitivity and specificity. Heterozygous showed an apparently reduced kinetic rate and a preserved secretory capacity. CONCLUSION: We tested a safe, well-tolerated needle-free image-based sweat test potentially applicable in children. Modelling responses by NLME allowed evaluating metrics of CFTR-dependent effects reflecting secretory capacity and kinetic rate.

On the Corner of Models and Cure: Gene Editing in Cystic Fibrosis.

Cystic fibrosis (CF) is a severe genetic disease for which curative treatment is still lacking. Next generation biotechnologies and more efficient cell-based and in vivo disease models are accelerating the development of novel therapies for CF. Gene editing tools, like CRISPR-based systems, can be used to make targeted modifications in the genome, allowing to correct mutations directly in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene. Alternatively, with these tools more relevant disease models can be generated, which in turn will be invaluable to evaluate novel gene editing-based therapies for CF. This critical review offers a comprehensive description of currently available tools for genome editing, and the cell and animal models which are available to evaluate them. Next, we will give an extensive overview of proof-of-concept applications of gene editing in the field of CF. Finally, we will touch upon the challenges that need to be addressed before these proof-of-concept studies can be translated towards a therapy for people with CF.

Aerosol-Mediated Non-Viral Lung Gene Therapy: The Potential of Aminoglycoside-Based Cationic Liposomes.

Aerosol lung gene therapy using non-viral delivery systems represents a credible therapeutic strategy for chronic respiratory diseases, such as cystic fibrosis (CF). Progress in CF clinical setting using the lipidic formulation GL67A has demonstrated the relevance of such a strategy while emphasizing the need for more potent gene transfer agents. In recent years, many novel non-viral gene delivery vehicles were proposed as potential alternatives to GL67 cationic lipid. However, they were usually evaluated using procedures difficult or even impossible to implement in clinical practice. In this study, a clinically-relevant administration protocol via aerosol in murine lungs was used to conduct a comparative study with GL67A. Diverse lipidic compounds were used to prepare a series of formulations inspired by the composition of GL67A. While some of these formulations were ineffective at transfecting murine lungs, others demonstrated modest-to-very-efficient activities and a series of structure-activity relationships were unveiled. Lipidic aminoglycoside derivative-based formulations were found to be at least as efficient as GL67A following aerosol delivery of a luciferase-encoding plasmid DNA. A single aerosol treatment with one such formulation was found to mediate long-term lung transgene expression, exceeding half the animal's lifetime. This study clearly supports the potential of aminoglycoside-based cationic lipids as potent GL67-alternative scaffolds for further enhanced aerosol non-viral lung gene therapy for diseases such as CF.

Vardenafil increases intracellular accumulation of the most prevalent mutant cystic fibrosis transmembrane conductance regulator (CTFR) in human bronchial epithelial cells.

Cystic fibrosis (CF) is a genetic disease characterized by progressive lung and chronic digestive manifestations. We have shown that therapeutic doses of vardenafil, a phosphodiesterase type 5 (PDE5) inhibitor, corrects CF Transmembrane conductance Regulator (CFTR)-dependent chloride transport in respiratory and intestinal tissues of F508del homozygous mice. Here, we studied the effect of vardenafil on CFTR in 16HBE14o- and CFBE41o- cell lines. First, the expression levels of PDE5 mRNA in these cell lines were monitored. The two cell lines were exposed to different drugs (dimethyl sulfoxide, 8-Br-cGMP, forskolin or vardenafil). The cAMP and cGMP intracellular concentrations were measured. Finally, we localised the CFTR by immunolabelling. PDE5 was similarly expressed in both wild-type and in CF cells. A fast and transient rise in cGMP intracellular contents followed treatment with vardenafil, confirming its PDE5 inhibitory effect. We showed that vardenafil promoted both the early steps of the cellular processing and the trafficking of F508del without fully addressing the protein to the plasma membrane. The effect was not reproduced by the brominated cGMP analogue and it was not prevented by the combination of a protein kinase G (PKG) inhibitor and vardenafil. These findings support the view that vardenafil partially rescues F508del through cGMP/PKG-independent mechanisms.

Antibacterial and transfection activities of nebulized formulations incorporating long n-alkyl chain silver N-heterocyclic carbene complexes.

The development of new antibacterial molecules is essential in view of the emergence of pathogenic strains resistant to multiple antibiotics. Among the infectious pathologies, pulmonary infections are particularly difficult to treat due to the complexity of lung anatomy and the presence of natural barriers such as mucus. At present, the aerosol delivery of antibacterial compounds is still poorly employed. Furthermore, the presence of bacteria in lungs negatively affects aerosolized Cystic Fibrosis gene therapy efficiency. A multi-functional formulation (antibacterial and transfection activities) could increase the therapeutic effect. This work reports the synthesis of new N-heterocyclic carbene silver complexes (Ag-NHC) featuring a lipid chain and the evaluation of their antibacterial potency, especially when delivered following aerosolization. When formulated alone in water, these Ag-NHC displayed remarkable antibacterial activities against some Staphyloccocus aureus strains and Pseudomonas aeruginosa clinical strains. Moreover, combined with cationic lipid and DNA (ternary combination), they could be used to deliver therapeutic genes via aerosolization in infected lungs. Altogether, the data reported herein support n-alkyl chain Ag-NHC as a possible alternative to conventional antibiotics for treating respiratory infections and to combat the emergence of multi-resistant bacteria.

Antibacterial effect and DNA delivery using a combination of an arsonium-containing lipophosphoramide with an N-heterocyclic carbene-silver complex - Potential benefits for cystic fibrosis lung gene therapy.

Cystic Fibrosis (CF), the most common chronic genetic disorder among the Caucasian population, is a life-threatening disease mainly due to respiratory failures resulting from chronic infections and inflammation. Although research in the pharmacological field has recently made significant progress, gene therapy still remains a promising strategy to cure CF, especially because it should be applicable to any patient whatever the mutation profile. Until now, little attention has been paid to bacterial lung infections with regard to gene delivery to the airways; yet, this could greatly impact on the success of gene therapy. Previously, we have reported arsonium-containing lipophosphoramides as poly-functional nanocarriers capable of simultaneous antibacterial action against Gram-positive bacteria and gene transfer into eukaryotic cells. In the present work, we show that such nanoparticles can also be combined with an N-heterocyclic carbene-silver complex in order to extend the spectrum of antibacterial activity, including towards the Gram-negative Pseudomonas aeruginosa. Importantly, this is demonstrated not only using standard in vitro protocols but also a clinically-relevant aerosol delivery method. Furthermore, antibacterial effects are compatible with efficient and safe gene delivery into human bronchial epithelial cells. The poly-functionality of combinations of such chemical compounds may thus show benefits for CF lung gene therapy.

Enhancement of lung gene delivery after aerosol: a new strategy using non-viral complexes with antibacterial properties.

The pathophysiology of obstructive pulmonary diseases, such as cystic fibrosis (CF), leads to the development of chronic infections in the respiratory tract. Thus, the symptomatic management of the disease requires, in particular, repetitive antibiotherapy. Besides these antibacterial treatments, certain pathologies, such as CF or chronic obstructive pulmonary disease (COPD), require the intake of many drugs. This simultaneous absorption may lead to undesirable drug interactions. For example, Orkambi® (lumacaftor/Ivacaftor, Vertex), a pharmacological drug employed to treat F508del patients, cannot be used with antibiotics such as rifampicin or rifabutin (rifamycin family) which are necessary to treat Mycobacteriaceae. As far as gene therapy is concerned, bacteria and/or biofilm in the airways present an additional barrier for gene transfer. Thus, aerosol administration of nanoparticles have to overcome many obstacles before allowing cellular penetration of therapeutic compounds. This review focusses on the development of aerosol formulations adapted to the respiratory tract and its multiple barriers. Then, formulations that are currently used in clinical applications are summarized depending on the active molecule delivered. Finally, we focus on new therapeutic approaches to reduce possible drug interactions by transferring the antibacterial activity to the nanocarrier while ensuring the transfection efficiency.