Chronic obstructive pulmonary disease (COPD) is a debilitating heterogeneous disease characterised by unregulated proteolytic destruction of lung tissue mediated via a protease-antiprotease imbalance. In COPD, the relationship between the neutrophil serine protease, neutrophil elastase, and its endogenous inhibitor, alpha-1-antitrypsin (AAT) is the best characterised. AAT belongs to a superfamily of serine protease inhibitors known as serpins. Advances in screening technologies have, however, resulted in many members of the serpin superfamily being identified as having differential expression across a multitude of chronic lung diseases compared to healthy individuals. Serpins exhibit a unique suicide-substrate mechanism of inhibition during which they undergo a dramatic conformational change to a more stable form. A limitation is that this also renders them susceptible to disease-causing mutations. Identification of the extent of their physiological/pathological role in the airways would allow further expansion of knowledge regarding the complexity of protease regulation in the lung and may provide wider opportunity for their use as therapeutics to aid the management of COPD and other chronic airways diseases.

Background Vascular endothelial growth factors (VEGFs) are key regulators of endothelial cell function and angiogenesis. We and others have previously demonstrated that VEGF-A stimulates AMP-activated protein kinase (AMPK) in cultured endothelial cells. Furthermore, AMPK has been reported to regulate VEGF-mediated angiogenesis. The role of AMPK in the function of VEGF-B remains undetermined, as does the role of AMPK in VEGF-stimulated endothelial cell proliferation, a critical process in angiogenesis. Methods Human aortic endothelial cells (HAECs) were incubated with VEGF-A and VEGF-B prior to examination of HAEC AMPK activity, proliferation, migration, fatty acid oxidation and fatty acid transport. The role of AMPK in the functional effects of VEGF-A and/or VEGF-B was assessed after downregulation of AMPK activity with chemical inhibitors or infection with adenoviruses expressing a dominant negative mutant AMPK. Results Incubation of HAECs with VEGF-B rapidly stimulated AMPK activity in a manner sensitive to an inhibitor of Ca 2+/calmodulin-dependent kinase kinase (CaMKK), without increasing phosphorylation of endothelial NO synthase (eNOS) phosphorylation at Ser1177. Downregulation of AMPK abrogated HAEC proliferation in response to VEGF-A or VEGF-B. However, activation of AMPK by agents other than VEGF inhibited proliferation. Downregulation of AMPK abrogated VEGF-A-stimulated HAEC migration, whereas infection with adenoviruses expressing constitutively active mutant AMPK stimulated chemokinesis. Neither VEGF-A nor VEGF-B had any significant effect on HAEC fatty acid oxidation, yet prolonged incubation with VEGF-A stimulated fatty acid uptake in an AMPK-dependent manner. Inhibition of eNOS abrogated VEGF-mediated proliferation and migration, but was without effect on VEGF-stimulated fatty acid transport, ERK or Akt phosphorylation. Conclusions These data suggest that VEGF-B stimulates AMPK by a CaMKK-dependent mechanism and stimulation of AMPK activity is required for proliferation in response to either VEGF-A or VEGF-B and migration in response to VEGF-A. AMPK activation alone was not sufficient, however, to stimulate proliferation in the absence of VEGF. VEGF-stimulated NO synthesis is required for the stimulation of proliferation by VEGF-A or VEGF-B, yet this may be independent of eNOS Ser1177 phosphorylation.

Common airborne allergens (pollen, animal dander and those from fungi and insects) are the main triggers of type I allergic disorder in the respiratory system and are associated with allergic rhinitis, allergic asthma, as well as immunoglobulin E (IgE)-mediated allergic bronchopulmonary aspergillosis. These allergens promote IgE crosslinking, vasodilation, infiltration of inflammatory cells, mucosal barrier dysfunction, extracellular matrix deposition and smooth muscle spasm, which collectively cause remodelling of the airways. Fungus and insect (house dust mite and cockroaches) indoor allergens are particularly rich in proteases. Indeed, more than 40 different types of aeroallergen proteases, which have both IgE-neutralising and tissue-destructive activities, have been documented in the Allergen Nomenclature database. Of all the inhaled protease allergens, 85% are classed as serine protease activities and include trypsin-like, chymotrypsin-like and collagenolytic serine proteases. In this article, we review and compare the allergenicity and proteolytic effect of allergen serine proteases as listed in the Allergen Nomenclature and MEROPS databases and highlight their contribution to allergic sensitisation, disruption of the epithelial barrier and activation of innate immunity in allergic airways disease. The utility of small-molecule inhibitors of allergen serine proteases as a potential treatment strategy for allergic airways disease will also be discussed.

Trypsin-like proteases (TLPs) belong to a family of serine enzymes with primary substrate specificities for the basic residues, lysine and arginine, in the P1 position. Whilst initially perceived as soluble enzymes that are extracellularly secreted, a number of novel TLPs that are anchored in the cell membrane have since been discovered. Muco-obstructive lung diseases (MucOLDs) are characterised by the accumulation of hyper-concentrated mucus in the small airways, leading to persistent inflammation, infection and dysregulated protease activity. Although neutrophilic serine proteases, particularly neutrophil elastase, have been implicated in the propagation of inflammation and local tissue destruction, it is likely that the serine TLPs also contribute to various disease-relevant processes given the roles that a number of these enzymes play in the activation of both the epithelial sodium channel (ENaC) and protease-activated receptor 2 (PAR2). More recently, significant attention has focused on the activation of viruses such as SARS-CoV-2 by host TLPs. The purpose of this review was to highlight key TLPs linked to the activation of ENaC and PAR2 and their association with airway dehydration and inflammatory signalling pathways, respectively. The role of TLPs in viral infectivity will also be discussed in the context of the inhibition of TLP activities and the potential of these proteases as therapeutic targets.

Cystic fibrosis (CF) is a life-limiting genetic disorder caused by loss-of-function mutations in the gene which codes for the CF transmembrane conductance regulator (CFTR) Cl⁻ channel. Loss of Cl⁻ secretion across the apical membrane of airway lining epithelial cells results in dehydration of the airway surface liquid (ASL) layer which impairs mucociliary clearance (MCC), and as a consequence promotes bacterial infection and inflammation of the airways. Interventions that restore airway hydration are known to improve MCC. Here we review the ion channels present at the luminal surface of airway epithelial cells that may be targeted to improve airway hydration and MCC in CF airways.

Many bacterial and viral pathogens (or their toxins), including Pseudomonas aeruginosa exotoxin A, require processing by host pro-protein convertases such as furin to cause disease. We report the development of a novel irreversible inhibitor of furin (QUB-F1) consisting of a diphenyl phosphonate electrophilic warhead coupled with a substrate-like peptide (RVKR), that also includes a biotin tag, to facilitate activity-based profiling/visualisation. QUB-F1 displays greater selectivity for furin, in comparison to a widely used exemplar compound (furin I) which has a chloromethylketone warhead coupled to RVKR, when tested against the serine trypsin-like proteases (trypsin, prostasin and matriptase), factor Xa and the cysteine protease cathepsin B. We demonstrate QUB-F1 does not prevent P. aeruginosa exotoxin A-induced airway epithelial cell toxicity; in contrast to furin I, despite inhibiting cell surface furin-like activity to a similar degree. This finding indicates additional proteases, which are sensitive to the more broad-spectrum furin I compound, may be involved in this process.