Heparan Sulfate is Shed into the Alveolus after Intratracheal Lung Injury and Facilitates Alveolar Epithelial Growth Factor Signaling and Lung Repair

Abstract

Heparan sulfate proteoglycans (HSPGs) are proteins covalently bound to heparan sulfate (HS), a linear highly‐sulfated polysaccharide, and are present in both the extracellular matrix and on cell surfaces. During tissue injury, HSPGs are thought to be shed from the injured cell surface; for example, the protein component of the cell membrane‐bound HSPG, syndecan‐1, has been shown to be shed from the respiratory epithelial cell surface into the airway during naphthalene lung injury. However, it remains unknown whether heparan sulfate (HS) is also shed into the airspace during intratracheal (IT) lung injury. Additionally, as HS is known to function as a scaffolding molecule for numerous growth factor ligands and receptors, facilitating ligand‐receptor binding and enhancing signaling, it is important to determine if HS is shed into the airspace during lung injury, as the shed HS may affect growth factor signaling and lung repair.To determine if HS is shed into the airspace during lung injury, we intratracheally instilled lipopolysaccharide (LPS) in mice and performed mass spectrometry for HS in bronchoalveolar (BAL) fluid collected from mice days 2, 4, 6, and 8 after IT LPS instillation. We detected increased BAL HS on days 2 (2.3 fold) and 4 (3.2 fold) after IT LPS instillation in comparison to animals instilled with PBS as control. To determine if the airspace HS enhances lung epithelial growth factor signaling and repair, we tested if exogenous HS enhances ERK signaling in alveolar type II (ATII) epithelial cells, progenitor cells that re‐epithelialize the alveolus after injury. We treated MLE‐12 cells (a mouse ATII cell line) with 0.5μg/ml HS and determined that, while HS inhibits ERK signaling in naïve MLE‐12 cells, HS permits ERK signaling in MLE‐12 cells that are enzymatically devoid of cell‐surface HS. This finding suggests that airspace HS may facilitate growth factor signaling in ATII cells that have shed their cell‐surface HS during injury. Finally, to determine if supplementation with exogenous HS enhances lung repair in vivo, we treated animals with intravenous HS on day 4 after IT LPS instillation and measured outcomes of lung injury. Twenty‐four hours after supplementation, HS reduced BAL total protein by 30%, indicating that exogenous HS supplementation may enhance alveolar epithelial barrier function and lung repair.Support or Funding InformationNHLBI RO1 HL125371