Henry Danahay

Notch2 Is Required for Inflammatory Cytokine-Driven Goblet Cell Metaplasia in the Lung

The balance and distribution of epithelial cell types is required to maintain tissue homeostasis. A hallmark of airway diseases is epithelial remodeling, leading to increased goblet cell numbers and an overproduction of mucus. In the conducting airway, basal cells act as progenitors for both secretory and ciliated cells. To identify mechanisms regulating basal cell fate, we developed a screenable 3D culture system of airway epithelial morphogenesis. We performed a high-throughput screen using a collection of secreted proteins and identified inflammatory cytokines that specifically biased basal cell differentiation toward a goblet cell fate, culminating in enhanced mucus production. We also demonstrate a specific requirement for Notch2 in cytokine-induced goblet cell metaplasia in vitro and in vivo. We conclude that inhibition of Notch2 prevents goblet cell metaplasia induced by a broad range of stimuli and propose Notch2 neutralization as a therapeutic strategy for preventing goblet cell metaplasia in airway diseases.

Camostat attenuates airway epithelial sodium channel function in vivo through the inhibition of a channel-activating protease

Inhibition of airway epithelial sodium channel (ENaC) function enhances mucociliary clearance (MCC). ENaC is positively regulated by channel-activating proteases (CAPs), and CAP inhibitors are therefore predicted to be beneficial in diseases associated with impaired MCC. The aims of the present study were to 1) identify low-molecular-weight inhibitors of airway CAPs and 2) to establish whether such CAP inhibitors would translate into a negative regulation of ENaC function in vivo, with a consequent enhancement of MCC. To this end, camostat, a trypsin-like protease inhibitor, provided a potent (IC50 ∼50 nM) and prolonged attenuation of ENaC function in human airway epithelial cell models that was reversible upon the addition of excess trypsin. In primary human bronchial epithelial cells, a potency order of placental bikunin > camostat > 4-guanidinobenzoic acid 4-carboxymethyl-phenyl ester > aprotinin >> soybean trypsin inhibitor = α1-antitrypsin, was largely consistent with that observed for inhibition of prostasin, a molecular candidate for the airway CAP. In vivo, topical airway administration of camostat induced a potent and prolonged attenuation of ENaC activity in the guinea pig trachea (ED50 = 3 μg/kg). When administered by aerosol inhalation in conscious sheep, camostat enhanced MCC out to at least 5 h after inhaled dosing. In summary, camostat attenuates ENaC function and enhances MCC, providing an opportunity for this approach toward the negative regulation of ENaC function to be tested therapeutically.

Active site conformational changes of prostasin provide a new mechanism of protease regulation by divalent cations.

Prostasin or human channel‐activating protease 1 has been reported to play a critical role in the regulation of extracellular sodium ion transport via its activation of the epithelial cell sodium channel. Here, the structure of the extracellular portion of the membrane associated serine protease has been solved to high resolution in complex with a nonselective d‐FFR chloromethyl ketone inhibitor, in an apo form, in a form where the apo crystal has been soaked with the covalent inhibitor camostat and in complex with the protein inhibitor aprotinin. It was also crystallized in the presence of the divalent cation Ca+2. Comparison of the structures with each other and with other members of the trypsin‐like serine protease family reveals unique structural features of prostasin and a large degree of conformational variation within specificity determining loops. Of particular interest is the S1 subsite loop which opens and closes in response to basic residues or divalent ions, directly binding Ca+2 cations. This induced fit active site provides a new possible mode of regulation of trypsin‐like proteases adapted in particular to extracellular regions with variable ionic concentrations such as the outer membrane layer of the epithelial cell.

Epithelial mucus-hypersecretion and respiratory disease.

Mucus production, secretion and clearance are considered to play a critical role in maintenance of airway health, however in diseases such as COPD, epidemiological and pathological studies suggest that excess mucus contributes to airway plugging and decline in lung health. The airway surface epithelium is composed of a heterogeneous mix of cell types one of which, the goblet cell, is dedicated to the production of secretory gel-forming mucins. Changes in epithelial cellular composition and function in response to irritants and microbes generally leads to enhanced co-ordinated functioning of the major facets of the mucociliary clearance (MCC) system i.e. mucus secretion, ion/fluid transport and ciliary function. The presence of mucus plugs in the airways of COPD patients demonstrates that facets of the MCC system have become compromised i.e. normally co-ordinated epithelial functions have become uncoupled. Almost nothing is known about the processes leading to such uncoupling. Understanding these processes may provide insights into mechanisms involved in regulation of epithelial integrity and the genesis of respiratory diseases such as COPD. In this review we will discuss regulation of airway epithelial cellular composition and function primarily with respect to goblet cell formation, mucus secretion, airway surface liquid (ASL) homeostasis, hydration of secreted mucus and ciliary clearance. We will discuss the functional overlap between cell populations, the potential impact of derivation from different progenitors and the implications of generating high goblet cell densities in the surface epithelium. The aim of this review is to stimulate discussion and develop hypotheses that could help to determine the mechanisms behind epithelial dysfunction in respiratory disease.

Discovery of a novel chemotype of potent human ENaC blockers using a bioisostere approach. Part 1: quaternary amines.

We report the identification of a novel series of human epithelial sodium channel (ENaC) blockers that are structurally distinct from the pyrazinoyl guanidine chemotype found in prototypical ENaC blockers such as amiloride. Following a rational design hypothesis a series of quaternary amines were prepared and evaluated for their ability to block ion transport via ENaC in human bronchial epithelial cells (HBECs). Compound 11 has an IC(50) of 200nM and is efficacious in the Guinea-pig tracheal potential difference (TPD) model of ENaC blockade with an ED(50) of 44μgkg(-1) at 1h. As such, pyrazinoyl quaternary amines represent the first examples of a promising new class of human ENaC blockers.

Development of primary human nasal epithelial cell cultures for the study of cystic fibrosis pathophysiology

Cultured primary epithelial cells are used to examine inflammation in cystic fibrosis (CF). We describe a new human model system using cultured nasal brushings. Nasal brushings were obtained from 16 F508del homozygous patients and 11 healthy controls. Cells were resuspended in airway epithelial growth medium and seeded onto collagen-coated flasks and membranes for use in patch-clamp, ion transport, and mediator release assays. Viable cultures were obtained with a 75% success rate from subjects with CF and 100% from control subjects. Amiloride-sensitive epithelial Na channel current of similar size was present in both cell types while forskolin-activated CF transmembrane conductance regulator current was lacking in CF cells. In Ussing chambers, cells from CF patients responded to UTP but not to forskolin. Spontaneous and cytomix-stimulated IL-8 release was similar (stimulated 29,448 ± 9,025 pg/ml; control 16,336 ± 3,308 pg/ml CF; means ± SE). Thus nasal epithelial cells from patients with CF can be grown from nasal brushings and used in electrophysiological and mediator release studies in CF research.

Respiratory Syncytial Virus Can Infect Basal Cells and Alter Human Airway Epithelial Differentiation

Respiratory syncytial virus (RSV) is a major cause of morbidity and mortality worldwide, causing severe respiratory illness in infants and immune compromised patients. The ciliated cells of the human airway epithelium have been considered to be the exclusive target of RSV, although recent data have suggested that basal cells, the progenitors for the conducting airway epithelium, may also become infected in vivo. Using either mechanical or chemical injury models, we have demonstrated a robust RSV infection of p63+ basal cells in air-liquid interface (ALI) cultures of human bronchial epithelial cells. In addition, proliferating basal cells in 2D culture were also susceptible to RSV infection. We therefore tested the hypothesis that RSV infection of this progenitor cell would influence the differentiation status of the airway epithelium. RSV infection of basal cells on the day of seeding (MOI≤0.0001), resulted in the formation of an epithelium that showed a profound loss of ciliated cells and gain of secretory cells as assessed by acetylated α-tubulin and MUC5AC/MUC5B immunostaining, respectively. The mechanism driving the switch in epithelial phenotype is in part driven by the induced type I and type III interferon response that we demonstrate is triggered early following RSV infection. Neutralization of this response attenuates the RSV-induced loss of ciliated cells. Together, these data show that through infection of proliferating airway basal cells, RSV has the potential to influence the cellular composition of the airway epithelium. The resulting phenotype might be expected to contribute towards both the severity of acute infection, as well as to the longer-term consequences of viral exacerbations in patients with pre-existing respiratory diseases.

Discovery of inhibitors of the channel-activating protease prostasin (CAP1/PRSS8) utilizing structure-based design.

Structure-based design was utilized to guide the early stage optimization of a substrate-like inhibitor to afford potent peptidomimetic inhibitors of the channel-activating protease prostasin. The first X-ray crystal structures of prostasin with small molecule inhibitors bound to the active site are also reported.

P2Y2-receptor-mediated activation of a contralateral, lanthanide-sensitive calcium entry pathway in the human airway epithelium.

Receptor‐mediated calcium entry (RMCE) was examined in well‐differentiated cultures of normal human bronchial epithelial cells (HBECs). Changes in intracellular free Ca2+ ([Ca2+]i) were quantified using fluorescence ratio imaging of Fura‐2‐loaded cells during perfusion with Ca2+ mobilizing agonists. Initial studies revealed an agonist potency of ATP=uridine triphosphate (UTP) >ADP=uridine diphosphate, consistent with purinergic activation of an apical P2Y2‐receptor mediating the increase in [Ca2+]i in HBECs. Apical UTP (30 μM) induced a sustained period of elevated [Ca2+]i between 300 and 600 s following agonist stimulation that extracellular Ca2+ free studies indicated was dominated by Ca2+ influx. RMCE was inhibited by 100 nM La3+ (83±3%) or Gd3+ (95±7%) (P<0.005, n=4–11) and was partially attenuated by Ni2+ (1 mM) (58.7±5.0%, P<0.005, n=9). RMCE was also partially sensitive (< 25% inhibition, P<0.01) to the cation channel blockers SKF96365 (30 μM) and econazole (30 μM), but was insensitive to both verapamil (1 μM) and ruthenium red (10 μM). Using either a sided Ca2+ readdition protocol or unilateral La3+, established that the RMCE pathway was located exclusively on the basolateral membrane. The pharmacological sensitivity of the P2Y2‐receptor activated Ca2+ entry pathway in the human airway epithelium is inconsistent with the established profile of TRP channel families and is therefore likely to be of an as‐yet uncharacterized molecular identity.

Reduced Sodium Transport With Nasal Administration of the Prostasin Inhibitor Camostat in Subjects With Cystic Fibrosis

BACKGROUND Prostasin, a trypsin-like serine protease, is a channel-activating protease and major regulator of epithelial sodium channel-mediated sodium absorption. Its direct inhibition by camostat represents a potential approach to inhibiting sodium transport in cystic fibrosis (CF). METHODS To determine whether a topical formulation of camostat represents an efficacious and tolerable approach to reducing Na+ transport in the CF airway, we conducted a two-part randomized, double-blind, placebo-controlled, crossover, ascending single-dose study to evaluate the pharmacodynamics, safety, and pharmacokinetics of camostat administered through a nasal spray pump in subjects with CF. Nasal potential difference (PD) was measured before and after treatment, and safety and pharmacokinetics were assessed by a standardized approach. RESULTS In part 1, nine subjects were enrolled, and six completed crossover dosing at the maximally tolerated dose. The change in maximal (most polarizing) basal PD 2 h following administration of camostat was +13.1 mV (1.6-mg dose group) compared with -8.6 mV following placebo (P<.005). Intrasubject change in Ringer and amiloride-sensitive PDs exhibited similar and consistent responses. Bayesian analysis in an additional six subjects in part 2 estimated a dose of 18 μg/mL to provide 50% of the maximum effect. There was no significant change in chloride transport or total nasal symptom score, nasal examination rating, and laboratory parameters. CONCLUSIONS This study establishes the proof of concept that a reduction in sodium transport in the human CF airway can be achieved through inhibition of prostasin activity, identifying a potential therapeutic target in the disease. TRIAL REGISTRATION ClinicalTrials.gov; No.: NCT00506792; URL: www.clinicaltrials.gov.