Role of the Epithelial Glycocalyx in Maintaining the Alveolar‐Capillary Barrier During Acute Lung Injury

Abstract

RationaleDisruption of the alveolar‐capillary barrier is a signature feature of acute lung injury. Previous studies have shown the importance of the glycocalyx in maintaining the integrity and homeostasis of the lung endothelium during acute lung injury. However, the role of the glycocalyx on lung epithelial barriers during acute lung injury has not been fully explored. We hypothesize that the lung epithelial glycocalyx is important in maintaining lung epithelial tight junctions in the setting of acute lung injury.MethodsCultured mouse lung epithelial cells (MLE12) were treated with functional or heat‐inactivated heparinase I/III. Additionally, some MLE12 cells were treated with LPS after pretreatment with functional heparinase I/III. Glycocalyx shedding was measured by immunohistochemistry for heparan sulfate (HS). To assess paracellular permeability, electrical cell‐impedance sensing (ECIS) was used to measure the resistance across the epithelial monolayer. To determine whether loss of the glycocalyx affects epithelial tight junctions we measured gene and protein expression of ZO‐1, by PCR, western blot, and immunofluorescence.ResultsMLE12 cells in culture express a robust glycocalyx that is completely shed following treatment with active heparinase I/III at 24 hours. Active heparinase I/III did not cause significant change in monolayer resistance from baseline compared to inactive heparinase I/III by ECIS (Active −142±39.2 v Inactive −118.9±57.9 ohms, p=0.75). Active heparinase I/III did not decrease protein expression of ZO‐1 compared to inactive heparinase I/III (Active 1.55±0.33 v Inactive 1.8±0.25 fold change ZO‐1/Actin, p=0.61). MLE12 cells treated with activated heparinase I/III and LPS had a significantly greater decrease in resistance from baseline compared to inactive heparinase and LPS (Figure , Active + LPS −145±8.8 vs Inactive + LPS − 7.7±10.8 ohms, p=0.0022)ConclusionTreating MLE12 cells with functional heparinase I/III degrades the epithelial glycocalyx. Loss of the glycocalyx alone was not sufficient to cause permeability defects nor alter the expression of tight junction proteins. However, glycocalyx shedding did increase the susceptibility of MLE12 cells to LPS‐induced injury. In clinical situations, the shedding of the glycocalyx in ALI is often accompanied by compounding factors such as infection, inflammation, or mechanical stress. The current findings suggest that damage to the epithelial glycocalyx may play a key role in maintaining the alveolar epithelial barrier in acute lung injury.Support or Funding InformationSupported by a research grant from Department of DefenseMLE12 cells treated with active heparinase + LPS had a significantly greater decrease in resistance compared to baseline than cells treated with inactive heparinase + LPS p=0.0022.Figure 1