scholarly journals Engineered mutant α-ENaC subunit mRNA delivered by lipid nanoparticles reduces amiloride currents in cystic fibrosis–based cell and mice models

2020 ◽  
Vol 6 (47) ◽  
pp. eabc5911
Author(s):  
Anindit Mukherjee ◽  
Kelvin D. MacDonald ◽  
Jeonghwan Kim ◽  
Michael I. Henderson ◽  
Yulia Eygeris ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Therapeutic Potential ◽  
Lipid Nanoparticles ◽  
Sodium Absorption ◽  
Airway Surface Liquid ◽  
Surface Liquid ◽  
Cf Transmembrane Conductance Regulator ◽  
Airway Cells

Cystic fibrosis (CF) results from mutations in the chloride-conducting CF transmembrane conductance regulator (CFTR) gene. Airway dehydration and impaired mucociliary clearance in CF is proposed to result in tonic epithelial sodium channel (ENaC) activity, which drives amiloride-sensitive electrogenic sodium absorption. Decreasing sodium absorption by inhibiting ENaC can reverse airway surface liquid dehydration. Here, we inhibit endogenous heterotrimeric ENaC channels by introducing inactivating mutant ENaC α mRNA (αmutENaC). Lipid nanoparticles carrying αmutENaC were transfected in CF-based airway cells in vitro and in vivo. We observed a significant decrease in macroscopic as well as amiloride-sensitive ENaC currents and an increase in airway surface liquid height in CF airway cells. Similarly, intranasal transfection of αmutENaC mRNA decreased amiloride-sensitive nasal potential difference in CFTRKO mice. These data suggest that mRNA-based ENaC inhibition is a powerful strategy for reducing mucus dehydration and has therapeutic potential for treating CF in all patients, independent of genotype.

Effect of Oral Digoxin, Topical Ouabain and Salbutamol on Transepithelial Nasal Potential Difference in Patients with Cystic Fibrosis

1995 ◽  
Vol 89 (3) ◽  
pp. 277-284 ◽  
Author(s):  
D. G. Peckham ◽  
A. Conn ◽  
C. Chotai ◽  
S. Lewis ◽  
A. J. Knox
Keyword(s):  
Cystic Fibrosis ◽  
Potential Difference ◽  
Sodium Absorption ◽  
Double Blind ◽  
Double Blind Placebo ◽  
Control Subjects ◽  
Healthy Control ◽  
Nasal Potential Difference

1. Airway epithelium in cystic fibrosis is characterized by a defect in chloride secretion across the apical membrane and an increase in sodium absorption. The increased rate of sodium absorption can be inhibited in vitro by ouabain, a Na+-K+-ATPase inhibitor, and in cystic fibrosis patients the number and activity of nasal epithelial Na+-K+-ATPase pumps is increased. 2. We have performed a series of studies to determine whether drugs which modify airway epithelial Na+-K+-ATPase activity in vitro can modify nasal potential in cystic fibrosis patients in vivo. As transepithelial nasal potential difference measurements were used to study the effect of drug modulation of airway epithelial ion transport in vivo, the repeatability of the technique was first evaluated. In order to assess the effectiveness of the technique used for measuring nasal potential difference, a pilot study was carried out using topical amiloride, a drug which has previously been shown to inhibit airway epithelium sodium transport in vivo. We then studied the effects of ouabain and digoxin, two inhibitors of Na+-K+-ATPase, and salbutamol, a drug which activates Na+-K+-ATPase, on nasal potential difference. 3. In study 1, nasal potential difference measurements were repeated on non-consecutive days in 20 patients with cystic fibrosis and 20 healthy individuals. Healthy subjects had a mean (SEM) potential difference value of −19.5 (0.9) mV, with a 95% range for a single estimate of 75–133%. In patients with cystic fibrosis, the mean (SEM) potential difference was −40.4 (2.1) mV, with a 95% range for a single estimate of 74–136%. 4. In an initial pilot study, the effect of topical amiloride on nasal potential difference was investigated on two consecutive days in four cystic fibrosis patients and four healthy control subjects, in a double-blind, placebo-controlled, randomized cross-over study. Nasal transepithelial potential was measured before and at 5, 15, 30, 45 and 60 min after the intranasal administration of 0.4 ml of a fine spray of 1 mmol/l amiloride or 0.9% saline placebo to both nostrils. Amiloride was associated with a maximal reduction in nasal potential difference at 15 min of 49% and 41% in cystic fibrosis patients and control subjects, respectively. Compared with saline, the amiloride response was significant in both groups (P < 0.025). 5. In study 2, the effect of topical ouabain and salbutamol on nasal potential difference was investigated in ten cystic fibrosis patients and ten healthy control subjects, in a double-blind, placebo-controlled, randomized cross-over study. Nasal transepithelial potential was measured before and at 5, 15, 30, 45 and 60 min after the intranasal administration of either 0.4 ml of a fine spray of 5 mg/ml salbutamol, 0.25 mg/ml ouabain or 0.9% saline placebo to both nostrils. There was no significant change in nasal potential difference with either ouabain, salbutamol or placebo in either healthy control subjects or patients with cystic fibrosis. 6. In study 3, we performed a randomized, double-blind, placebo-controlled cross-over study of oral digoxin on nasal potential difference, spirometry and sweat electrolytes for 2 weeks in 11 patients with cystic fibrosis. During the treatment period, patients had a mean (range) serum digoxin level after the first and second week of therapy of 0.9 (0.3–1.4) μg/l and 1.1 (0.4–2.2) μg/l, respectively. There was no significant difference in the change in nasal potential difference measurements, forced expiratory volume in 1 s and sweat Na/Cl concentrations between the digoxin and placebo trial periods. 7. In conclusion, neither topical ouabain nor systemic digoxin had any effect on nasal potential difference in cystic fibrosis. Inhibitors of Na+-K+-ATPase are therefore unlikely to find a role in the treatment of cystic fibrosis. The lack of a detrimental effect of salbutamol on nasal potential difference is reassuring, as β-agonists are widely used in patients with cystic fibrosis.

Bioelectric characteristics of exon 10 insertional cystic fibrosis mouse: comparison with humans

1995 ◽  
Vol 268 (2) ◽  
pp. C297-C307 ◽  
Author(s):  
S. N. Smith ◽  
D. M. Steel ◽  
P. G. Middleton ◽  
F. M. Munkonge ◽  
D. M. Geddes ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Intestinal Tract ◽  
Chloride Secretion ◽  
Sodium Absorption ◽  
Wild Type ◽  
Nasal Potential Difference ◽  
New Treatments ◽  
Exon 10

Two important issues that can be addressed by animal models are disease pathogenesis and the testing of new treatments, including gene therapy. How closely these models mimic the relevant disorder in humans will determine their usefulness. This study examines how closely the characteristic bioelectric features of cystic fibrosis (CF) are reproduced in the airways and intestinal tract of the exon 10 insertional mutant mouse (cf/cf). In agreement with CF subjects these cf/cf mutant mice demonstrate the following: 1) reduced adenosine 3',5'-cyclic monophosphate-related chloride secretion throughout the respiratory and intestinal tracts both in vivo and in vitro, 2) calcium-related chloride secretion that is preserved in the airways but reduced in the intestine, and 3) a more negative nasal potential difference and increased amiloride response compared with wild-type animals, likely to relate to increased sodium absorption. In contrast to humans, sodium absorption is not increased in the small intestine and is reduced in the trachea of the cf/cf mice. We conclude that the majority of the salient electrophysiological features of CF required for studies of pathogenesis or testing of new treatments are present in these cf/cf mice.

scholarly journals Antimicrobial Properties of Mesenchymal Stem Cells: Therapeutic Potential for Cystic Fibrosis Infection, and Treatment

2016 ◽  
Vol 2016 ◽  
pp. 1-12 ◽  
Author(s):  
Morgan T. Sutton ◽  
David Fletcher ◽  
Santosh K. Ghosh ◽  
Aaron Weinberg ◽  
Rolf van Heeckeren ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
Bacterial Growth ◽  
Therapeutic Potential ◽  
Bioactive Molecules ◽  
Human Mscs ◽  
Antimicrobial Effectiveness

Cystic fibrosis (CF) is a genetic disease in which the battle between pulmonary infection and inflammation becomes the major cause of morbidity and mortality. We have previously shown that human MSCs (hMSCs) decrease inflammation and infection in thein vivomurine model of CF. The studies in this paper focus on the specificity of the hMSC antimicrobial effectiveness usingPseudomonas aeruginosa(gram negative bacteria) andStaphylococcus aureus(gram positive bacteria). Our studies show that hMSCs secrete bioactive molecules which are antimicrobialin vitroagainstPseudomonas aeruginosa, Staphylococcus aureus,andStreptococcus pneumonia, impacting the rate of bacterial growth and transition into colony forming units regardless of the pathogen. Further, we show that the hMSCs have the capacity to enhance antibiotic sensitivity, improving the capacity to kill bacteria. We present data which suggests that the antimicrobial effectiveness is associated with the capacity to slow bacterial growth and the ability of the hMSCs to secrete the antimicrobial peptide LL-37. Lastly, our studies demonstrate that the tissue origin of the hMSCs (bone marrow or adipose tissue derived), the presence of functional cystic fibrosis transmembrane conductance regulator (CFTR: human,Cftr: mouse) activity, and response to effector cytokines can impact both hMSC phenotype and antimicrobial potency and efficacy. These studies demonstrate, the unique capacity of the hMSCs to manage different pathogens and the significance of their phenotype in both the antimicrobial and antibiotic enhancing activities.

scholarly journals Culture with apically applied healthy or disease sputum alters the airway surface liquid proteome and ion transport across human bronchial epithelial cells.

2021 ◽  
Author(s):  
Maximillian Woodall ◽  
Boris Reidel ◽  
Mehmet Kesimer ◽  
Robert Tarran ◽  
Deborah L Baines
Keyword(s):  
Epithelial Cells ◽  
Ion Transport ◽  
Short Circuit ◽  
Bronchial Epithelial Cells ◽  
Airway Surface Liquid ◽  
Human Bronchial Epithelial Cells ◽  
Bronchial Epithelial ◽  
Surface Liquid

Airway secretions contain many signalling molecules and peptides/proteins that are not found in airway surface liquid (ASL) generated by normal human bronchial epithelial cells (NHBE) in vitro. These play a key role in innate defence and mediate communication between the epithelium, immune cells and the external environment. We investigated how culture of NHBE with apically applied secretions from healthy or disease (Cystic Fibrosis, CF) lungs affected epithelial function with a view to providing better in vitro models of the in vivo environment. NHBE from 6-8 different donors were cultured at air-liquid interface (ALI), with apically applied sputum from normal healthy donors (NLS) or CF donors (CFS) for 2-4 hours, 48 hours or with sputum reapplied over 48 hours. Proteomic analysis was carried out on the sputa and on NHBE ASL before and after culture with sputa. Transepithelial electrical resistance (TEER), short circuit current (Isc) and changes to ASL height were measured. There were 71 proteins common to both sputa but not ASL. The protease:protease inhibitor balance was increased in CFS compared to NLS and ASL. Culture of NHBE with sputa for 48 hours identified additional factors not present in NLS, CFS or ASL alone. Culture with either NLS or CFS for 48 hours increased CFTR activity, calcium activated chloride channel (CaCC) activity and changed ASL height. These data indicate that culture with healthy or disease sputum changes the proteomic profile of ASL and ion transport properties of NHBE and this may increase physiological relevance when using in vitro airway models.

scholarly journals Determination of rheology and surface tension of airway surface liquid: a review of clinical relevance and measurement techniques

2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Zhenglong Chen ◽  
Ming Zhong ◽  
Yuzhou Luo ◽  
Linhong Deng ◽  
Zhaoyan Hu ◽  
...  
Keyword(s):  
Surface Tension ◽  
Clinical Relevance ◽  
Measurement Techniques ◽  
Barrier Properties ◽  
Airway Surface Liquid ◽  
Surfactant Replacement Therapy ◽  
Surface Liquid ◽  
The Stability

AbstractBy airway surface liquid, we mean a thin fluid continuum consisting of the airway lining layer and the alveolar lining layer, which not only serves as a protective barrier against foreign particles but also contributes to maintaining normal respiratory mechanics. In recent years, measurements of the rheological properties of airway surface liquid have attracted considerable clinical attention due to new advances in microrheology instruments and methods. This article reviews the clinical relevance of measurements of airway surface liquid viscoelasticity and surface tension from four main aspects: maintaining the stability of the airways and alveoli, preventing ventilator-induced lung injury, optimizing surfactant replacement therapy for respiratory syndrome distress, and characterizing the barrier properties of airway mucus to improve drug and gene delivery. Primary measuring techniques and methods suitable for determining the viscoelasticity and surface tension of airway surface liquid are then introduced with respect to principles, advantages and limitations. Cone and plate viscometers and particle tracking microrheometers are the most commonly used instruments for measuring the bulk viscosity and microviscosity of airway surface liquid, respectively, and pendant drop methods are particularly suitable for the measurement of airway surface liquid surface tension in vitro. Currently, in vivo and in situ measurements of the viscoelasticity and surface tension of the airway surface liquid in humans still presents many challenges.

scholarly journals In Vivo Airway Surface Liquid Cl− Analysis with Solid-State Electrodes

2001 ◽  
Vol 119 (1) ◽  
pp. 3-14 ◽  
Author(s):  
Ray A. Caldwell ◽  
Barbara R. Grubb ◽  
Robert Tarran ◽  
Richard C. Boucher ◽  
Michael R. Knowles ◽  
...  
Keyword(s):  
Solid State ◽  
Airway Surface Liquid ◽  
Culture Models ◽  
Normal Human ◽  
Surface Liquid ◽  
Multiple Species ◽  
Cell Culture Models ◽  
Made In

The pathogenesis of cystic fibrosis (CF) airways disease remains controversial. Hypotheses that link mutations in CFTR and defects in ion transport to CF lung disease predict that alterations in airway surface liquid (ASL) isotonic volume, or ion composition, are critically important. ASL [Cl−] is pivotal in discriminating between these hypotheses, but there is no consensus on this value given the difficulty in measuring [Cl−] in the “thin” ASL (∼30 μm) in vivo. Consequently, a miniaturized solid-state electrode with a shallow depth of immersion was constructed to measure ASL [Cl−] in vivo. In initial experiments, the electrode measured [Cl−] in physiologic salt solutions, small volume (7.6 μl) test solutions, and in in vitro cell culture models, with ≥93% accuracy. Based on discrepancies in reported values and/or absence of data, ASL Cl− measurements were made in the following airway regions and species. First, ASL [Cl−] was measured in normal human nasal cavity and averaged 117.3 ± 11.2 mM (n = 6). Second, ASL [Cl−] measured in large airway (tracheobronchial) regions were as follows: rabbit trachea and bronchus = 114.3 ± 1.8 mM; (n = 6) and 126.9 ± 1.7 mM; (n = 3), respectively; mouse trachea = 112.8 ± 4.2 mM (n = 13); and monkey bronchus = 112.3 ± 10.9 mM (n = 3). Third, Cl− measurements were made in small (1–2 mm) diameter airways of the rabbit (108.3 ± 7.1 mM, n = 5) and monkey (128.5 ± 6.8 mM, n = 3). The measured [Cl−], in excess of 100 mM throughout all airway regions tested in multiple species, is consistent with the isotonic volume hypothesis to describe ASL physiology.

scholarly journals Cooh-Terminal Truncations Promote Proteasome-Dependent Degradation of Mature Cystic Fibrosis Transmembrane Conductance Regulator from Post-Golgi Compartments

2001 ◽  
Vol 153 (5) ◽  
pp. 957-970 ◽  
Author(s):  
Mohamed Benharouga ◽  
Martin Haardt ◽  
Norbert Kartner ◽  
Gergely L. Lukacs
Keyword(s):  
Cystic Fibrosis ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Cell Surface Biotinylation ◽  
Partially Unfolded ◽  
Cf Transmembrane Conductance Regulator

Impaired biosynthetic processing of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR), a cAMP-regulated chloride channel, constitutes the most common cause of CF. Recently, we have identified a distinct category of mutation, caused by premature stop codons and frameshift mutations, which manifests in diminished expression of COOH-terminally truncated CFTR at the cell surface. Although the biosynthetic processing and plasma membrane targeting of truncated CFTRs are preserved, the turnover of the complex-glycosylated mutant is sixfold faster than its wild-type (wt) counterpart. Destabilization of the truncated CFTR coincides with its enhanced susceptibility to proteasome-dependent degradation from post-Golgi compartments globally, and the plasma membrane specifically, determined by pulse–chase analysis in conjunction with cell surface biotinylation. Proteolytic cleavage of the full-length complex-glycosylated wt and degradation intermediates derived from both T70 and wt CFTR requires endolysosomal proteases. The enhanced protease sensitivity in vitro and the decreased thermostability of the complex-glycosylated T70 CFTR in vivo suggest that structural destabilization may account for the increased proteasome susceptibility and the short residence time at the cell surface. These in turn are responsible, at least in part, for the phenotypic manifestation of CF. We propose that the proteasome-ubiquitin pathway may be involved in the peripheral quality control of other, partially unfolded membrane proteins as well.

Role of airway surface liquid and submucosal glands in cystic fibrosis lung disease

2003 ◽  
Vol 284 (1) ◽  
pp. C2-C15 ◽  
Author(s):  
A. S. Verkman ◽  
Yuanlin Song ◽  
Jay R. Thiagarajah
Keyword(s):  
Cystic Fibrosis ◽  
Lung Disease ◽  
Model Systems ◽  
Airway Surface Liquid ◽  
Cystic Fibrosis Lung Disease ◽  
Submucosal Glands ◽  
Cftr Protein ◽  
Surface Liquid ◽  
Gland Function ◽  
Cf Transmembrane Conductance Regulator

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) protein, an epithelial chloride channel expressed in the airways, pancreas, testis, and other tissues. A central question is how defective CFTR function in CF leads to chronic lung infection and deterioration of lung function. Several mechanisms have been proposed to explain lung disease in CF, including abnormal airway surface liquid (ASL) properties, defective airway submucosal gland function, altered inflammatory response, defective organellar acidification, loss of CFTR regulation of plasma membrane ion transporters, and others. This review focuses on the physiology of the ASL and submucosal glands with regard to their proposed role in CF lung disease. Experimental evidence for defective ASL properties and gland function in CF is reviewed, and deficiencies in understanding ASL/gland physiology are identified as areas for further investigation. New model systems and measurement technologies are being developed to make progress in establishing lung disease mechanisms in CF, which should facilitate mechanism-based design of therapies for CF.

scholarly journals Mathematical model reveals role of nucleotide signaling in airway surface liquid homeostasis and its dysregulation in cystic fibrosis

2017 ◽  
Vol 114 (35) ◽  
pp. E7272-E7281 ◽  
Author(s):  
Conner I. Sandefur ◽  
Richard C. Boucher ◽  
Timothy C. Elston
Keyword(s):  
Mathematical Model ◽  
Cystic Fibrosis ◽  
Ion Transport ◽  
Purinergic Signaling ◽  
Nucleotide Metabolism ◽  
Sodium Absorption ◽  
Ciliary Activity ◽  
Airway Surface Liquid ◽  
Surface Hydration ◽  
Surface Liquid

Mucociliary clearance is composed of three components (i.e., mucin secretion, airway surface hydration, and ciliary-activity) which function coordinately to clear inhaled microbes and other foreign particles from airway surfaces. Airway surface hydration is maintained by water fluxes driven predominantly by active chloride and sodium ion transport. The ion channels that mediate electrogenic ion transport are regulated by extracellular purinergic signals that signal through G protein-coupled receptors. These purinoreceptors and the signaling pathways they activate have been identified as possible therapeutic targets for treating lung disease. A systems-level description of airway surface liquid (ASL) homeostasis could accelerate development of such therapies. Accordingly, we developed a mathematical model to describe the dynamic coupling of ion and water transport to extracellular purinergic signaling. We trained our model from steady-state and time-dependent experimental measurements made using normal and cystic fibrosis (CF) cultured human airway epithelium. To reproduce CF conditions, reduced chloride secretion, increased potassium secretion, and increased sodium absorption were required. The model accurately predicted ASL height under basal normal and CF conditions and the collapse of surface hydration due to the accelerated nucleotide metabolism associated with CF exacerbations. Finally, the model predicted a therapeutic strategy to deliver nucleotide receptor agonists to effectively rehydrate the ASL of CF airways.

scholarly journals Thickness of the airway surface liquid layer in the lung is affected in cystic fibrosis by compromised synergistic regulation of the ENaC ion channel

2019 ◽  
Vol 16 (157) ◽  
pp. 20190187 ◽  
Author(s):  
Daniel V. Olivença ◽  
Luis L. Fonseca ◽  
Eberhard O. Voit ◽  
Francisco R. Pinto
Keyword(s):  
Cystic Fibrosis ◽  
Steady State ◽  
Phosphoinositide Metabolism ◽  
Airway Surface Liquid ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Therapeutic Benefits ◽  
Synergistic Regulation ◽  
Surface Liquid ◽  
Cf Transmembrane Conductance Regulator

The lung epithelium is lined with a layer of airway surface liquid (ASL) that is crucial for healthy lung function. ASL thickness is controlled by two ion channels: epithelium sodium channel (ENaC) and cystic fibrosis (CF) transmembrane conductance regulator (CFTR). Here, we present a minimal mathematical model of ENaC, CFTR and ASL regulation that sheds light on the control of ENaC by the short palate lung and nasal epithelial clone 1 (SPLUNC1) protein and by phosphatidylinositol 4,5-biphosphate (PI(4,5)P 2 ). The model, despite its simplicity, yields a good fit to experimental observations and is an effective tool for exploring the interplay between ENaC, CFTR and ASL. Steady-state data and dynamic information constrain the model's parameters without ambiguities. Testing the hypothesis that PI(4,5)P 2 protects ENaC from ubiquitination suggests that this protection does not improve the model results and that the control of the ENaC opening probability by PI(4,5)P 2 is sufficient to explain all available data. The model analysis further demonstrates that ASL at the steady state is sensitive to small changes in PI(4,5)P 2 abundance, particularly in CF conditions, which suggests that manipulation of phosphoinositide metabolism may promote therapeutic benefits for CF patients.

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