Barbara Dhooghe

Cystic fibrosis: insight into CFTR pathophysiology and pharmacotherapy

Cystic fibrosis is the most common life-threatening recessively inherited disease in Caucasians. Due to early provision of care in specialized reference centers and more comprehensive care, survival has improved over time. Despite great advances in supportive care and in our understanding of its pathophysiology, there is still no cure for the disease. Therapeutic strategies aimed at rescuing the abnormal protein are either being sought after or under investigation. This review highlights salient insights into pathophysiology and candidate molecules suitable for CFTR pharmacotherapy. Clinical trials using Ataluren, VX-809 and ivacaftor have provided encouraging data. Preclinical data with inhibitors of phosphodiesterase type 5, such as sildenafil and analogs, have highlighted their potential for CFTR pharmacotherapy. Because sildenafil and analogs are in clinical use for other clinical applications, research on this class of drugs might speed up the development of new therapies for CF.

Lung inflammation in cystic fibrosis: Pathogenesis and novel therapies

Despite remarkable progress following the identification of the causing gene, the final outcome of cystic fibrosis (CF) remains determined mainly by the progressive reduction of lung function. Inflammation of the airways is one of the key elements of the pathogenesis of the disease: it is responsible for the destruction of lung architecture, resulting in progressive loss of respiratory function. Bronchial infection induces an intense inflammatory reaction characterized by a massive invasion of neutrophils, the properties of which seems altered in CF. Moreover, the inflammatory process is also marked by a profuse release of soluble pro-inflammatory mediators, such as interleukin (IL)-6, IL-1β and IL-8 cytokines. In contrast, release of the anti-inflammatory mediator IL-10 is reduced, thus reflecting a pro-/anti-inflammatory imbalance. The inflammation/infection pair seems hard to dissociate, and the origin of the baneful consequences of the persisting excessive inflammatory responses remains to be cleared up: does inflammation follow or rather precede infection? Recent data suggest that uncontrolled inflammation is constitutive in CF. Countering it at early stages of the disease in order to prevent irretrievable damages in lungs remains a major priority in treating patients with CF. In this review, we discuss the usefulness and limitations of mouse models of CF to study the pathogenesis of human lung inflammatory disease, and the development of new potential strategies to reduce the inflammatory burden in the airways.

Immunomodulatory activity of vardenafil on induced lung inflammation in cystic fibrosis mice

BACKGROUND We tested the hypothesis that vardenafil, a common drug used for improving erectile dysfunction and able to partially normalize transepithelial chloride transport in cystic fibrosis (CF), modulates CF lung inflammation. METHODS Inflammatory markers in lungs of F508del-CF and wild-type mice were monitored in response to lipopolysaccharide from Pseudomonas aeruginosa (LPS). The effect of pretreatment with vardenafil (0.14 mg/kg) was evaluated. RESULTS A latent inflammatory status, characterized by neutrophil infiltrate, mouse macrophage inflammatory protein (MIP)-2 and tumor necrosis factor (TNF)-α, was found in baseline conditions in F508del-CF mice. Inflammatory markers were increased after LPS with higher responses in CF. Vardenafil globally attenuated inflammatory responses in both genotypes however reduction of macrophage infiltration, macrophage chemoattractant chemokine and interleukin-1β was observed in the CF group only. CONCLUSION Vardenafil reduces lung inflammation with a more pronounced effect in F508del-CF mice, particularly on macrophage cell markers.

Dysregulated Proinflammatory and Fibrogenic Phenotype of Fibroblasts in Cystic Fibrosis

Morbi-mortality in cystic fibrosis (CF) is mainly related to chronic lung infection and inflammation, uncontrolled tissue rearrangements and fibrosis, and yet the underlying mechanisms remain largely unknown. We evaluated inflammatory and fibrosis responses to bleomycin in F508del homozygous and wild-type mice, and phenotype of fibroblasts explanted from mouse lungs and skin. The effect of vardenafil, a cGMP-specific phosphodiesterase type 5 inhibitor, was tested in vivo and in culture. Responses of proinflammatory and fibrotic markers to bleomycin were enhanced in lungs and skin of CF mice and were prevented by treatment with vardenafil. Purified lung and skin fibroblasts from CF mice proliferated and differentiated into myofibroblasts more prominently and displayed higher sensitivity to growth factors than those recovered from wild-type littermates. Under inflammatory stimulation, mRNA and protein expression of proinflammatory mediators were higher in CF than in wild-type fibroblasts, in which CFTR expression reached similar levels to those observed in other non-epithelial cells, such as macrophages. Increased proinflammatory responses in CF fibroblasts were reduced by half with submicromolar concentrations of vardenafil. Proinflammatory and fibrogenic functions of fibroblasts are upregulated in CF and are reduced by vardenafil. This study provides compelling new support for targeting cGMP signaling pathway in CF pharmacotherapy.

PDE5 Inhibitors as Potential Tools in the Treatment of Cystic Fibrosis.

Despite great advances in the understanding of the genetics and pathophysiology of cystic fibrosis (CF), there is still no cure for the disease. Using phosphodiesterase type 5 (PDE5) inhibitors, we and others have provided evidence of rescued F508del-CFTR trafficking and corrected deficient chloride transport activity. Studies using PDE5 inhibitors in mice homozygous for the clinically relevant F508del mutation have been conducted with the aim of restoring F508del-CFTR protein function. We demonstrated, by measuring transepithelial nasal potential difference in F508del mice following intraperitoneal injection of sildenafil, vardenafil, or taladafil at clinical doses are able to restore the decreased CFTR-dependent chloride transport across the nasal mucosa. Moreover, vardenafil, but not sildenafil, stimulates chloride transport through the normal CFTR protein. We developed a specific nebulizer setup for mice, with which we demonstrated, through a single inhalation of PDE5 inhibitors, local activation of CFTR protein in CF. Significant potential advantages of inhalation drug therapy over oral or intravenous routes include rapid onset of pharmacological action, reduced systemic secondary effects, and reduced effective drug doses compared to the drug delivered orally; this underlines the relevance and impact of our work for translational science. More recently, we analyzed the bronchoalveolar lavage of CF and wild-type mice for cell infiltrates and expression of pro-inflammatory cytokines and chemokines; we found that the CFTR activating effect of vardenafil, selected as a representative long-lasting PDE5 inhibitor, breaks the vicious circle of lung inflammation which plays a major role in morbi-mortality in CF. Our data highlight the potential use of PDE5 inhibitors in CF. Therapeutic approaches using clinically approved PDE5 inhibitors to address F508del-CFTR defects could speed up the development of new therapies for CF.

Strategies in early clinical development for the treatment of basic defects of cystic fibrosis

ABSTRACT Introduction: Twenty-six years after the identification of the gene responsible for cystic fibrosis (CF), controversies still surround the pathogenesis of the disease that continues to burden and shorten lives. Therefore, finding effective therapeutic strategies that target the basic defect of CF is crucially needed. Areas covered: This review offers a comprehensive survey of fundamental therapies in early stages of development for the treatment of CF. The first part describes recent strategies targeting the basic defect either at the gene or at the transcript level. The second part summarizes a panel of novel strategies targeting protein repair. The third part reports strategies targeting non-CFTR channels. Expert opinion: Recent major breakthroughs in CF therapy have been made, raising hope to find a cure for CF. Apart from Vertex corrector and potentiator molecules (lumacaftor, ivacaftor, VX-661) and from ataluren, used to correct nonsense mutations, most compounds being currently tested are in very early (I-II) phases of development and definitive clinical results are keenly expected. Among the broad list of molecules and strategies being tested, the QR-010 compound and inhibitors of phosphodiesterase type 5 (sildenafil, vardenafil) could reveal a strong potentiality as therapeutic candidates to cure CF.

Correction of chloride transport and mislocalization of CFTR protein by vardenafil in the gastrointestinal tract of cystic fibrosis mice.

Although lung disease is the major cause of mortality in cystic fibrosis (CF), gastrointestinal (GI) manifestations are the first hallmarks in 15–20% of affected newborns presenting with meconium ileus, and remain major causes of morbidity throughout life. We have previously shown that cGMP-dependent phosphodiesterase type 5 (PDE5) inhibitors rescue defective CF Transmembrane conductance Regulator (CFTR)-dependent chloride transport across the mouse CF nasal mucosa. Using F508del-CF mice, we examined the transrectal potential difference 1 hour after intraperitoneal injection of the PDE5 inhibitor vardenafil or saline to assess the amiloride-sensitive sodium transport and the chloride gradient and forskolin-dependent chloride transport across the GI tract. In the same conditions, we performed immunohistostaining studies in distal colon to investigate CFTR expression and localization. F508del-CF mice displayed increased sodium transport and reduced chloride transport compared to their wild-type littermates. Vardenafil, applied at a human therapeutic dose (0.14 mg/kg) used to treat erectile dysfunction, increased chloride transport in F508del-CF mice. No effect on sodium transport was detected. In crypt colonocytes of wild-type mice, the immunofluorescence CFTR signal was mostly detected in the apical cell compartment. In F508del-CF mice, a 25% reduced signal was observed, located mostly in the subapical region. Vardenafil increased the peak of intensity of the fluorescence CFTR signal in F508del-CF mice and displaced it towards the apical cell compartment. Our findings point out the intestinal mucosa as a valuable tissue to study CFTR transport function and localization and to evaluate efficacy of therapeutic strategies in CF. From our data we conclude that vardenafil mediates potentiation of the CFTR chloride channel and corrects mislocalization of the mutant protein. The study provides compelling support for targeting the cGMP signaling pathway in CF pharmacotherapy.

Resveratrol increases F508del-CFTR dependent salivary secretion in cystic fibrosis mice.

ABSTRACT Cystic fibrosis (CF) is a fatal genetic disease associated with widespread exocrine gland dysfunction. Studies have suggested activating effects of resveratrol, a naturally-occurring polyphenol compound with antioxidant and anti-inflammatory properties, on CF transmembrane conductance regulator (CFTR) protein function. We assayed, in F508del-CFTR homozygous (CF) and in wild-type mice, the effect of resveratrol on salivary secretion in basal conditions, in response to inhibition by atropine (basal β-adrenergic-dependent component) and to stimulation by isoprenaline (CFTR-dependent component). Both components of the salivary secretion were smaller in CF mice than in controls. Two hours after intraperitoneal administration of resveratrol (50 mg/kg) dissolved in DMSO, the compound was detected in salivary glands. As in both CF and in wild-type mice, DMSO alone increased the response to isoprenaline in males but not in females, the effect of resveratrol was only measured in females. In wild-type mice, isoprenaline increased secretion by more than half. In CF mice, resveratrol rescued the response to isoprenaline, eliciting a 2.5-fold increase of β-adrenergic-stimulated secretion. We conclude that the salivary secretion assay is suitable to test DMSO-soluble CFTR modulators in female mice. We show that resveratrol applied in vivo to mice reaches salivary glands and increases β-adrenergic secretion. Immunolabelling of CFTR in human bronchial epithelial cells suggests that the effect is associated with increased CFTR protein expression. Our data support the view that resveratrol is beneficial for treating CF. The salivary secretion assay has a potential application to test efficacy of novel CF therapies.

Azithromycin Attenuates Pseudomonas-Induced Lung Inflammation by Targeting Bacterial Proteins Secreted in the Cultured Medium

Background Pseudomonas aeruginosa airway infections are a major cause of morbidity and mortality in patients with cystic fibrosis (CF). Azithromycin improves the related clinical outcomes, but its mechanisms of action remain poorly understood. We tested the hypothesis that azithromycin downregulates P. aeruginosa-induced pro-inflammatory responses by modifying release of bacterial proteins. Methods We monitored inflammatory markers in lungs of CF mutant mice and their littermate controls in response to conditioned media (CM) collected from the reference P. aeruginosa PAO1 strain cultured in the presence or in the absence of azithromycin. A mass spectrometry-based proteomic approach was applied to examine whether the macrolide elicits a differential release of bacterial proteins. Results CM collected from azithromycin-untreated PAO1 cultures induced powerful pro-inflammatory neutrophil-dominated responses. Azithromycin attenuated the responses, mainly of macrophage chemoattractant protein-1, tumor necrosis factor-α, and interferon-γ, in CF but not in wild-type mice. Proteomic analysis showed that azithromycin upregulated an array of bacterial proteins including those associated with regulation of immune functions and with repair and resolution of inflammatory responses like the chaperone DnaK and the S-adenosylmethionine synthase, while it downregulated the extracellular heme acquisition protein HasA and the catalytic enzyme lysylendopeptidase. Conclusion Supernatants collected from cultures of the bacterial strain PAO1 represent a novel experimental model to trigger in vivo lung inflammatory responses that should be closer to those obtained with live bacteria, but without bacterial infection. Combined with a bactericidal effect, complex regulation of bacterial innate immune and metabolic factors released in the cultured medium by the action of the macrolide can contribute to its anti-inflammatory effects.

35 Deficiency of the Planar Cell Polarity protein CELSR3 in CF bronchial epithelial cells increases susceptibility to epithelial–mesenchymal transition

Objectives: Epithelial barrier function is impaired in CF airways as an indirect consequence of F508del-CFTR trafficking defect. The role of apico-basal polarity is crucial in the process but little is known about the influence of Planar Cell Polarity (PCP) which coordinates alignment of cell polarity across the tissue plane. Methods: Expression of several PCP proteins including atypical cadherin CELSR3 (Cadherin EGF LAG seven-pass G-type receptor 3) were quantified in normal and CF primary human bronchial epithelial (HBE) cells and HBE cell lines. To characterize the role of CELSR3 in epithelial polarity, CELSR3, epithelial and mesenchymal markers expression were quantified after inducing epithelial– mesenchymal transition (EMT) in HBEs monolayers with TGFb or cisplatin. To determine if CELSR3, suspected to undergo homophilic interactions to establish cell–cell interactions, is a key determinant of epithelial barrier function, transepithelial electric resistance (TEER) was measured after silencing CELSR3 expression in 16HBEs. Results: HBEs expressed several PCP proteins, including CELSR3 for which expression was downregulated in CF-HBEs compared to non-CF cells. Both TGFb and cisplatin induced concomitant loss of CELSR3 and epithelial marker ZO-1 and gain of mesenchymal (i.e. profibrotic) markers vimentin and fibronectin. CELSR3 siRNA in 16HBE cells enhanced TGFb-induced EMT. However, CELSR3 siRNA did not modulate TEER, indicating that CELSR3 is an epithelial marker but not a direct determinant of intercellular junction tightness. Conclusion: CELSR3 is an epithelial marker down-regulated in CF-HBEs. Loss of CELSR3 function may confer a profibrotic phenotype to CF-HBEs cells.