A glycopolymer improves vascoelasticity and mucociliary transport of abnormal cystic fibrosis mucus
Courtney M. Fernandez-Petty, … , Shenda M. Baker, Steven M. Rowe
Courtney M. Fernandez-Petty, … , Shenda M. Baker, Steven M. Rowe
Published April 18, 2019
Citation Information: JCI Insight. 2019;4(8):e125954. https://doi.org/10.1172/jci.insight.125954.
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Research Article Genetics Pulmonology

A glycopolymer improves vascoelasticity and mucociliary transport of abnormal cystic fibrosis mucus

Abstract

Cystic fibrosis (CF) is characterized by increased mucus viscosity and delayed mucociliary clearance that contributes to progressive decline of lung function. Mucus in the respiratory and GI tract is excessively adhesive in the presence of airway dehydration and excess extracellular Ca2+ upon mucin release, promoting hyperviscous, densely packed mucins characteristic of CF. Therapies that target mucins directly through ionic interactions remain unexploited. Here we show that poly (acetyl, arginyl) glucosamine (PAAG), a polycationic biopolymer suitable for human use, interacts directly with mucins in a Ca2+-sensitive manner to reduce CF mucus viscoelasticity and improve its transport. Notably, PAAG induced a linear structure of purified MUC5B and altered its sedimentation profile and viscosity, indicative of proper mucin expansion. In vivo, PAAG nebulization improved mucociliary transport in CF rats with delayed mucus clearance, and cleared mucus plugging in CF ferrets. This study demonstrates the potential use of a synthetic glycopolymer PAAG as a molecular agent that could benefit patients with a broad array of mucus diseases.

Authors

Courtney M. Fernandez-Petty, Gareth W. Hughes, Hannah L. Bowers, John D. Watson, Bradley H. Rosen, Stacy M. Townsend, Carlo Santos, Caroline E. Ridley, Kengyeh K. Chu, Susan E. Birket, Yao Li, Hui Min Leung, Marina Mazur, Bryan A. Garcia, T. Idil Apak Evans, Emily Falk Libby, Heather Hathorne, Justin Hanes, Guillermo J. Tearney, John P. Clancy, John F. Engelhardt, William E. Swords, David J. Thornton, William P. Wiesmann, Shenda M. Baker, Steven M. Rowe

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Figure 1

Effect of PAAG on the viscoelastic properties of expectorated CF sputum and mucus from cultured epithelial cells.

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Effect of PAAG on the viscoelastic properties of expectorated CF sputum ...
(A–E) Sputum collected from CF subjects was treated with PBS, PAAG, HPMC, DNase, or DTT as a positive control. (A) Dynamic viscosity (η) curves show decreased viscosity with PAAG at low (i.e., physiologic) frequency as compared with PBS. (B) Shear-dependent elastic modulus (G′) shows an effect of 100 μg/ml PAAG across all frequencies reduction in sputum elasticity compared with control. (C) Summary data of dynamic viscosity (η) at a frequency sweep of 0.8 Hz (low but reliable frequency, as expected to occur in vivo) shows a significant decrease in viscosity with 100 μg/ml PAAG treatment. Each line represents a sputum sample; some donors were evaluated more than once on separate clinical encounters (n = 13). (D) Similar analysis of sputum elastic modulus (G′). *P < 0.05 by Wilcoxon’s rank test. (E) Summary data of dynamic viscosity (η) at a frequency sweep at 0.8 Hz comparing the effect of PAAG with the addition of the neutral polymer control HPMC, the known mucolytic DNase, and the Ca2+ chelator EGTA. Replicates derived from n = 6 sputum samples and divided among conditions; *P < 0.05, **P < 0.01 by Kruskal-Wallis test with Dunn’s MCT. (F) Representative particle track of 500 nm PEG-coated nanoparticle placed in mucus layer of Phe508del/Phe508del HBE cells treated with PAAG (10 μl of 2, 5, and 10 mg/ml PAAG added apically, to achieve 100, 250, and 500 μg/ml final concentrations, respectively, in the ASL) versus PBS control 24 hours prior to assay. (G) Summary data plotting mean squared displacement (MSD) versus time. Increased slope is an indicator of increased diffusion and decreased mucus viscosity. Doses shown are estimated ASL concentrations, as described above. (H) Particle tracking microrheology (PTM) analysis of data in G (250 and 500 μg/ml) showed a marked decrease in viscosity as compared with vehicle control. Reduced viscosity was seen across all ranges of a frequency sweep (0.46–17.36 Hz), indicating that the effect was robust. Viscosity of normal HBE cells at 1.0 Hz is 17.7 ± 4.3 cP (23). (I) Summary data of frequency sweep at 1 Hz. (J) Summary data at frequency sweep 1 Hz with PAAG compared with the mucolytic DNase. n = 4 filters/condition. *P < 0.05, **P < 0.01, ***P < 0.001, #P = 0.11 by 2-way ANOVA with Tukey’s (G and H) or by Kruskal-Wallis test with Dunn’s MCT (I and J). (K and I) Fluorescence image of WT HBE and CF HBE monolayer treated with 250 μg/ml of PAAG-FITC shows that PAAG intermingles within the mucus layer (K) and is predominantly localized within close proximity to the epithelial surface (L). ASL dyed with Texas red to provide contrast. Scale bar: 10 μm; n = 4 filters/condition.