Abstract 15352: Lung Endothelial Transport in Glypican 1 Knockout Mice

Abstract

Introduction: Pulmonary edema is a life-threatening condition that can result from the acute increase in hemodynamic forces in the lung vasculature. The mechanisms whereby pulmonary edema develops are unresolved but increased lung endothelial permeability may be important. The endothelial glycocalyx is a surface layer known to mediate the effects of mechanical forces in the lung vasculature by eNOS-dependent mechanisms. Among the components of the glycocalyx, glypican 1 (Gpc1) is considered a potential mechano-transducer due to its physical interaction with the cellular membrane. Herein we sought to investigate the mechanisms whereby Gpc1 regulates pulmonary permeability. Hypothesis: We hypothesize that Gpc1 regulates endothelial transport by eNOS-dependent mechanisms. Methods: Mouse lung endothelial cells (MLEC) isolated from male and female wild-type (WT) and Gpc1 knockout mice (Gpc1KO, 6-8 weeks old) were used. Endothelial transport was assessed by dextran permeability in vitro, lung barrier stability was assessed by TEER, eNOS pathway was assessed by immunoblotting. To assess the role of eNOS to Gpc1 effects on endothelial transport, MLEC were exposed to an eNOS decoy peptide. Results: Gpc1KO MLEC formed a tighter barrier in vitro when compared to WT MLEC (3.18±0.11 [Gpc1KO] vs 2.34±0.11 [WT], normalized TEER, n=3/group). This was preserved during high pressure conditions. High pressure increased transcellular but not paracellular transport in both WT and Gpc1KO MLEC (1.8 fold in WT and 1.2 fold in Gpc1KO, n=6/group). Gpc1KO showed decreased eNOS expression (0.3 fold, n=4/group ) and activity during (1.947±0.32 [WT] vs 1.316±0.13 [Gpc1KO], n=4/group). The eNOS decoy peptide increased transcellular transport only in Gpc1KO MLEC (3 fold, n=5/group). Conclusions: In conclusion, these data suggest that intracellular transport may be the main mechanism for pressure-induced changes in endothelial permeability in MLEC . The mechanisms whereby Gpc1 regulates transcellular transport seem to be associated with eNOS signaling. This may be important for the management of acute changes in pressure seen in clinical settings when NO donors are used as vasodilators to decrease blood pressure.