Experimental Degradation of the Alveolar Epithelial Glycocalyx Decreases Lung Compliance in Young and Old Mice, Potentially by Inducing Surfactant Dysfunction

Abstract

Background The alveolar airspace is lined by a heparan sulfate (HS)-enriched epithelial glycocalyx. We previously demonstrated that direct lung injury (i.e., caused by inhalational insults) causes shedding of epithelial HS into the airspaces of mice and humans. However, the homeostatic function of epithelial surface HS is uncertain. We hypothesized that during lung homeostasis, epithelial HS contributes to the aqueous hypophase upon which surfactant is distributed. As such, experimental degradation of epithelial HS should lead to alveolar collapse and loss of lung compliance, key characteristics of ARDS pathophysiology. Methods and results: We treated young (8-10 week) and old (>22 month) C57BL/6 mice with 15 units intratracheal heparinase I/III (Hep I/III), a HS-specific glucuronidase that specifically degrades epithelial surface HS. Control mice received equal concentrations of heat-inactivated Hep I/III (HI-Hep I/III). 24 hours later, Hep I/III-treated mice demonstrated loss of inspiratory capacity (0.6114 ml vs. 0.7158 ml, n = 10 per group; p < 0.05) and loss of lung compliance (0.049 ml/cm H2O vs. 0.059 ml/cm H2O, n = 10, p < 0.05); similar results were seen in aged mice (1.061 ml vs. 1.170 ml; 0.082 ml/cm H2O vs 0.093 ml/cm H2O; n =3-4, p < 0.05 for both). Surprisingly, this rapid loss of compliance occurred in the absence of lung edema (as measured by wet/dry ratio and histology), fibrosis (histology), or inflammation (RNA sequencing, BAL cell count and differential). Micro-CT scans of the lungs of Hep I/III-treated young mice at increasing PEEP confirmed a loss of lung compliance and an increase in radiographic lung density (Hounsfield units). Preliminary lung stereology demonstrated the presence of microatelectasis, suggesting that loss of airspace HS could induce surfactant dysfunction. As BAL phospholipid analyses showed no differences in phospholipid species between Hep I/III- and HI-Hep I/III young mice, loss of airspace HS may cause functional impairments in surfactant, as opposed to alterations in surfactant expression. Conclusions Our findings suggest that loss of alveolar epithelial HS disrupts surfactant function, potentially via disrupting the epithelial aqueous hypophase. Reconstitution of epithelial glycocalyx integrity after ARDS would therefore be essential to lung repair and the subsequent return to homeostasis.