rapid-viscoelastic-characterization-airway-mucus-using-benchtop

Rapid Viscoelastic Characterization of Airway Mucus Using a Benchtop Rheometer

In muco-obstructive lung diseases (e.g., asthma, chronic obstructive pulmonary disease, cystic fibrosis) and other respiratory conditions (e.g., ...

The University of North Carolina at Chapel Hill

The epithelial sodium channel (ENaC) constitutes the rate-limiting step for sodium absorption across airway epithelia, which in turn regulates airway surface liquid (ASL) volume and the efficiency of mucociliary clearance. This role in ASL volume regulation suggests that ENaC activity is influenced by local factors rather than systemic signals indicative of total body volume homeostasis. Based on reports that ENaC may be regulated by extracellular serine protease activity in Xenopus and mouse renal epithelia, we sought to identify proteases that serve similar functions in human airway epithelia. Homology screening of a human airway epithelial cDNA library identified two trypsin-like serine proteases (prostasin and TMPRSS2) that, as revealed by in situ hybridization, are expressed in airway epithelia. Functional studies in the Xenopus oocyte expression system demonstrated that prostasin increased ENaC currents 60--80%, whereas TMPRSS2 markedly decreased ENaC currents and protein levels. Studies of primary nasal epithelial cultures in Ussing chambers revealed that inhibition of endogenous serine protease activity with aprotinin markedly decreased ENaC-mediated currents and sensitized the epithelia to subsequent channel activation by exogenous trypsin. These data, therefore, suggest that protease-mediated regulation of sodium absorption is a function of human airway epithelia, and prostasin is a likely candidate for this activity.

Regulation of the epithelial sodium channel by serine proteases in human airways.

5'-Nucleoside triphosphates (NTP) are present in the liquid covering airway surfaces and mediate important physiologic events through their interaction with P2-nucleotide receptors. Activation of airway P2Y(2) receptors, for example, stimulates ciliary beat frequency, chloride/liquid secretion, and goblet cell degranulation. We, therefore, have studied the metabolic pathways that regulate the concentration of nucleotides on airway surfaces. Stimulation of submucosal gland secretion in the nose was previously found to decrease the concentration of 5'-adenosine triphosphate (ATP) in nasal lavage samples due to the presence of a secreted 5'-nucleoside triphosphatase (NTPase). In this study, gland secretions were further studied and found to also contain adenylate kinase (AK) and nucleoside diphosphokinase (NDPK) activities. Ecto-AK and ecto-NDPK activities were also detected in well-differentiated cultures of superficial nasal epithelia, which reflected a combination of cell-associated and released (into culture media) AK and NDPK activities. This study demonstrates that "ecto-kinases" on airway surfaces (1) emanate from different enzyme families, including both AK and NDPK; (2) are expressed at superficial epithelial surfaces and in submucosal glands; and (3) may be important regulators of nucleotide concentrations on airway surfaces.

Secreted and cell-associated adenylate kinase and nucleoside diphosphokinase contribute to extracellular nucleotide metabolism on human airway surfaces.

It has been an ongoing challenge to translate knowledge pertaining to the molecular basis of cystic fibrosis (CF) into a clear understanding of the development of CF lung disease. Various hypotheses have attempted to explain the apparent breach of innate defenses in CF, although a definitive explanation has been elusive.

Update on pathogenesis of cystic fibrosis lung disease.

Abnormal homeostasis of the volume of airway surface liquid in patients with cystic fibrosis is thought to produce defects in mucus clearance and airway defense. Through osmotic forces, hypertonic saline may increase the volume of airway surface liquid, restore mucus clearance, and improve lung function.

Mucus clearance and lung function in cystic fibrosis with hypertonic saline.

Current concepts of cystic fibrosis (CF) pathophysiology link ion transport abnormalities to reduced airway surface liquid (ASL) hydration and impaired mucus clearance. It is likely that correction of the defects that cause ASL dehydration will prevent degradation of mucus clearance, thereby preventing the initiation and/or progression of CF lung disease. A number of novel therapeutic agents aimed at the earliest steps in disease pathogenesis are now under development for the treatment of CF lung disease. Consequently, there is a tremendous need to develop methods that directly assess the effects of these agents on the underlying pathophysiologic process in the target organ. The measurement of mucociliary clearance (MCC) is a highly biologically relevant outcome, but one that is in need of further development. Here, we describe important methodologic aspects of MCC measurement and issues that have limited its use as an outcome measure in the past. Furthermore, we outline the steps that are being carried out now, and will be carried out in the future, to improve the performance of these studies in clinical trials. A systematic approach to optimizing and standardizing the measurement of MCC should greatly advance our ability to assess novel therapies at a relatively early stage of drug development. The resulting data may then be used to select those candidates that should be rapidly advanced into larger clinical trials.

Mucociliary clearance as an outcome measure for cystic fibrosis clinical research.

The epithelial sodium channel (ENaC) represents the rate-limiting step of sodium absorption across airway epithelia and thereby constitutes the major pathway for volume absorption from the airway surface liquid compartment. ENaC dysregulation leads to dehydration of airway surfaces in patients with cystic fibrosis, which in turn disrupts the primary innate lung defense mechanism, mucus clearance. The development of treatment strategies that address this defect is a logical and promising means of preventing or delaying the onset of this lethal lung disease.

Sodium channels and cystic fibrosis.

Our current understanding of the pathogenesis of cystic fibrosis (CF) lung disease stresses the importance of the physical and chemical properties of the airway surface liquid (ASL). In particular, the loss of cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel function in CF reduces the volume and fluidity of the ASL, thus impairing mucociliary clearance and innate antimicrobial mechanisms. Besides direct approaches to restoring mutant CFTR function, alternative therapeutic strategies may also be considered to correct the basic defect of impaired salt and water transport. Such alternative strategies are focused on the restoration of mucociliary transport by (1) reducing sodium and fluid absorption by inhibiting the ENaC channel; (2) activating alternative chloride channels; and (3) increasing airway surface hydration with osmotic agents. Therapeutic approaches directed at targets other than CFTR are attractive because they are potentially useful to all patients irrespective of their genotype. Clinical trials are underway to test the efficacy of these approaches.

New pulmonary therapies directed at targets other than CFTR.

In human airways diseases, including cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD), host defense is compromised and airways inflammation and infection often result. Mucus clearance and trapping of inhaled pathogens constitute key elements of host defense. Clearance rates are governed by mucus viscous and elastic moduli at physiological driving frequencies, whereas transport of trapped pathogens in mucus layers is governed by diffusivity. There is a clear need for simple and effective clinical biomarkers of airways disease that correlate with these properties. We tested the hypothesis that mucus solids concentration, indexed as weight percent solids (wt%), is such a biomarker. Passive microbead rheology was employed to determine both diffusive and viscoelastic properties of mucus harvested from human bronchial epithelial (HBE) cultures. Guided by sputum from healthy (1.5-2.5 wt%) and diseased (COPD, CF; 5 wt%) subjects, mucus samples were generated in vitro to mimic in vivo physiology, including intermediate range wt% to represent disease progression. Analyses of microbead datasets showed mucus diffusive properties and viscoelastic moduli scale robustly with wt%. Importantly, prominent changes in both biophysical properties arose at ∼4 wt%, consistent with a gel transition (from a more viscous-dominated solution to a more elastic-dominated gel). These findings have significant implications for: (1) penetration of cilia into the mucus layer and effectiveness of mucus transport; and (2) diffusion vs. immobilization of micro-scale particles relevant to mucus barrier properties. These data provide compelling evidence for mucus solids concentration as a baseline clinical biomarker of mucus barrier and clearance functions. 

A biophysical basis for mucus solids concentration as a candidate biomarker for airways disease.

Airway dehydration in cystic fibrosis (CF) leads to chronic inflammation, ongoing infection and progressive lung disease. Restoration of airway hydration by inhalation of an osmotic agent (hypertonic saline) has been shown to be safe, effective and well-tolerated in adults with CF. Although the safety of hypertonic saline in infants and young children with CF has also been established, recent studies have reported inconclusive evidence about its efficacy. In this editorial, we discuss the evidence behind hypertonic saline use for adults, children and infants with CF.

Hypertonic saline for cystic fibrosis: worth its salt?

Cavosonstat (N91115), an orally bioavailable inhibitor of S-nitrosoglutathione reductase, promotes cystic fibrosis transmembrane conductance regulator (CFTR) maturation and plasma membrane stability, with a mechanism of action complementary to CFTR correctors and potentiators.

Pharmacokinetics and safety of cavosonstat (N91115) in healthy and cystic fibrosis adults homozygous for F508DEL-CFTR.

Inhaled hypertonic saline (HS) has been shown to increase mucociliary clearance (MCC) and improve clinical outcomes in adults and adolescents with cystic fibrosis (CF). However, in younger children with CF, a large study failed to demonstrate clinical benefits. This discrepancy could reflect pharmacodynamic differences in the MCC response to HS in different populations. We previously demonstrated the absence of a sustained effect of HS on MCC in healthy adults and in this study sought to characterize the durability of the MCC response to HS in adults with CF.

Hypertonic saline has a prolonged effect on mucociliary clearance in adults with cystic fibrosis.

Exercising our options: comparing effects of exercise and positive expiratory pressure on mucociliary clearance.

Hypertonic saline (HS) is commonly prescribed for children with cystic fibrosis (CF) despite the absence of strong data indicating clinical efficacy in a population with mild lung disease. We hypothesized that HS treatment would result in a sustained improvement in mucociliary clearance (MCC) in children with CF who had minimal lung disease, thus providing evidence for a biologically relevant effect that also may be associated with clinical improvements.

A four week trial of hypertonic saline in children with mild cystic fibrosis lung disease: Effect on mucociliary clearance and clinical outcomes.

CFTR function is required for normal mucociliary clearance (MCC) and cough-assisted clearance (CC). Lumacaftor-ivacaftor is approved for use in people with cystic fibrosis (CF) carrying two copies of F508del-CFTR. In this observational study performed at four study sites, we characterized the effect of lumacaftor-ivacaftor on mucociliary and cough clearance and related this to other clinical and research endpoints after one month of treatment. Twenty-five adolescents and adults were enrolled. No effect on whole lung MCC was observed, but CC was significantly increased. Sweat chloride improved by 18 mEq/L in this group, indicating a modest restoration of CFTR activity, but no demonstrable change in FEV or lung clearance index was observed. We speculate that the modest effect of lumacaftor-ivacaftor on CFTR function was insufficient to yield an improvement in MCC.

Effect of lumacaftor-ivacaftor on mucociliary clearance and clinical outcomes in cystic fibrosis: Results from the PROSPECT MCC sub-study.

 Advance care planning (ACP) is recommended for all patients with cystic fibrosis (CF), yet clear implementation guidelines do not exist.  The University of North Carolina Adult CF Care Team developed a process to implement semistructured multidisciplinary outpatient ACP meetings as routine care for patients with CF. Premeeting and post-meeting surveys were used to elicit patients' attitudes toward ACP.  Twenty-seven adults with CF completed a face-to-face ACP meeting, and 13 completed both surveys. Following the multidisciplinary ACP meeting, overall scores for understanding of ACP topics improved by 4.5 points ( = 0.003).  We successfully implemented sustainable ACP meetings for adults with CF and found increased comfort with ACP and documentation of wishes after ACP meetings. It is important for CF care providers to meet the needs of this patient population by ensuring that ACP is in place before crisis situations.

Article	Year
Regulation of the epithelial sodium channel by serine proteases in human airways. The Journal of biological chemistry\| PubMed ID: 11756432	2002
Secreted and cell-associated adenylate kinase and nucleoside diphosphokinase contribute to extracellular nucleotide metabolism on human airway surfaces. American journal of respiratory cell and molecular biology\| PubMed ID: 11804872	2002
Update on pathogenesis of cystic fibrosis lung disease. Current opinion in pulmonary medicine\| PubMed ID: 14534400	2003
Mucus clearance and lung function in cystic fibrosis with hypertonic saline. The New England journal of medicine\| PubMed ID: 16421365	2006
Mucociliary clearance as an outcome measure for cystic fibrosis clinical research. Proceedings of the American Thoracic Society\| PubMed ID: 17652507	2007
Sodium channels and cystic fibrosis. Chest\| PubMed ID: 17998363	2007
New pulmonary therapies directed at targets other than CFTR. Cold Spring Harbor perspectives in medicine\| PubMed ID: 23732851	2013
A biophysical basis for mucus solids concentration as a candidate biomarker for airways disease. PloS one\| PubMed ID: 24558372	2014
Hypertonic saline for cystic fibrosis: worth its salt? Expert review of respiratory medicine\| PubMed ID: 24666113	2014
Pharmacokinetics and safety of cavosonstat (N91115) in healthy and cystic fibrosis adults homozygous for F508DEL-CFTR. Journal of cystic fibrosis : official journal of the European Cystic Fibrosis Society\| PubMed ID: 28209466	2017
Hypertonic saline has a prolonged effect on mucociliary clearance in adults with cystic fibrosis. Journal of cystic fibrosis : official journal of the European Cystic Fibrosis Society\| PubMed ID: 29371133	2018
Exercising our options: comparing effects of exercise and positive expiratory pressure on mucociliary clearance. The European respiratory journal\| PubMed ID: 31000670	2019
A four week trial of hypertonic saline in children with mild cystic fibrosis lung disease: Effect on mucociliary clearance and clinical outcomes. Journal of cystic fibrosis : official journal of the European Cystic Fibrosis Society\| PubMed ID: 32669217	2020
Effect of lumacaftor-ivacaftor on mucociliary clearance and clinical outcomes in cystic fibrosis: Results from the PROSPECT MCC sub-study. Journal of cystic fibrosis : official journal of the European Cystic Fibrosis Society\| PubMed ID: 34083123	2022
Development of an Advance Care Planning Protocol in a Cystic Fibrosis Outpatient Clinic. Journal of palliative medicine\| PubMed ID: 34191614	2021

Scott H. Donaldson

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