2-O, 3-O-Desulfated Heparin Inhibits Neutrophil Elastase–Induced HMGB-1 Secretion and Airway Inflammation

Abstract

Neutrophil elastase (NE) is a major inflammatory mediator in cystic fibrosis (CF) that is a robust predictor of lung disease progression. NE directly causes airway injury via protease activity, and propagates persistent neutrophilic inflammation by upregulation of neutrophil chemokine expression. Despite its key role in the pathogenesis of CF lung disease, there are currently no effective antiprotease therapies available to patients with CF. Although heparin is an effective antiprotease and anti-inflammatory agent, its anticoagulant activity prohibits its use in CF, due to risk of pulmonary hemorrhage. In this report, we demonstrate the efficacy of a 2-O, 3-Odesulfated heparin (ODSH), a modified heparin with minimal anticoagulant activity, to inhibit NE activity and to block NE-induced airway inflammation. Using an established murine model of intratracheal NE-induced airway inflammation, we tested the efficacy of intratracheal ODSH to block NE-generated neutrophil chemoattractants and NE-triggered airway neutrophilic inflammation. ODSH inhibited NE-induced keratinocyte-derived chemoattractant and high-mobility group box 1 release in bronchoalveolar lavage. ODSH also blocked NE-stimulated highmobility group box 1 release from murine macrophages in vitro, and inhibited NE activity in functional assays consistent with prior reports of antiprotease activity. In summary, this report suggests that ODSH is a promising antiprotease and anti-inflammatory agent that may be useful as an airway therapy in CF.

2-O, 3-O Desulfated Heparin Blocks High Mobility Group Box 1 Release by Inhibition of p300 Acetyltransferase Activity

“…secretion into cell culture media. Importantly, we demonstrated that ODSH blocks NE release of HMGB1, at least in part, by inhibiting NE activity (17). In this report, we tested whether ODSH inhibited HMGB1 release from RAW264.7 cells when triggered by NE or by LPS, an inflammatory mediator.…”

“…Classically, HMGB1 is either secreted by macrophages and other airway cells after infection (11), inflammation (17), and oxidative stress (33,34), or released from necrotic cells, attracting neutrophils to necrotic tissues (35). Neither of the inflammatory stimuli used to activate HMGB1 release from RAW264.7 cells caused macrophage cell death, so HMGB1 was released by signaling mechanisms, resulting in secretion.…”

“…Loss of the 2-O and 3-O sulfate residues markedly reduces the anticoagulant activity of ODSH, which endows ODSH with a better safety profile as a therapeutic agent than its parent compound heparin for patients with CF. ODSH blocks HMGB1 in vivo in both a neutrophil elastase (NE)-induced intratracheal inflammatory model (17) and a Pseudomonas aeruginosa pneumonia model (18). However, the mechanism of how ODSH blocks HMGB1 secretion is unknown.…”

“…Western Analyses for HMGB1 and Acetyllysine HMGB1 Western analysis was performed as reported previously (17). For acetyllysine detection, recombinant HMGB1 was collected by Dynabeads (10103; Thermo Fisher, Waltham, MA), separated by 4-20% polyacrylamide gel electrophoresis, and transferred to nitrocellulose.…”

High-Mobility Group Box 1 Mediates Epithelial-to-Mesenchymal Transition in Pulmonary Fibrosis Involving Transforming Growth Factor-β1/Smad2/3 Signaling

“…Therefore, strategies disrupting TGF-b1/Smad signaling undoubtedly have therapeutic potential in the clinical treatment of PF. High-mobility group box 1 (HMGB1) is a transcription factor-like protein that acts as a danger signal in inflammatory diseases, tissue injury, as well as fibrotic diseases (Rowe et al, 2008;Ebina et al, 2011;Ogiku et al, 2011;Lee et al, 2013;Griffin et al, 2014;Li et al, 2014a). In lung tissues from patients with PF, HMGB1 is predominantly expressed in alveolar macrophages, infiltrating inflammatory cells, and epithelial cells (Hamada et al, 2008).…”