Glypican 1 mechanosensing mediates eNOS uncoupling during hydrostatic pulmonary edema

Abstract

Hydrostatic pulmonary edema is a life-threatening condition caused by an acute increase in pulmonary capillary pressure. The molecular mechanisms whereby hydrostatic pulmonary edema develops are unresolved. The pulmonary endothelial glycocalyx is a mechano-sensitive signaling layer known to regulate lung endothelial permeability. Within the glycocalyx, membrane-bound heparan sulfate proteoglycans (HSPGs) are putative mechano-sensors. Herein, we investigated if the membrane-bound HSPG glypican 1 is a mechanosensor in the lung vasculature and its role in hydrostatic pulmonary edema progression. Using an isolated perfused lung system, we showed that glypican 1 knockout mice (Gpc1^−/−) are protected from pressure-induced lung edema, a phenotype associated with impaired 70 KDa dextran transport and decreased reactive oxygen species (ROS) production. Using wild-type (WT) mouse lung endothelial cells (MLEC) and human lung microvascular endothelial cells (HLMEC), we show that high pressure induces the activation of Protein Kinase C-alpha (PKCα) at Y⁶⁵⁷, which phosphorylates endothelial nitric oxide synthase (eNOS) at T⁴⁹⁵. This is associated with increased ROS production by eNOS-dependent pathways. The inhibition of eNOS with ethyl thiourea (ETU) or N5-(1-iminoethyl)-l-ornithine (L-NIO) mitigates the effects of high pressure on ROS production, lung edema, and barrier stability. This pathologic signaling axis is not activated in Gpc1^−/− MLEC exposed to high-pressure conditions. Notably, cells deficient in Glypican 1 show increased phosphorylation of PKCα at T⁶³⁸, a site associated with PKCα stability and inactivation. The protective signaling mechanisms observed in Gpc1^−/− MLEC are replicated in HLMEC silenced for glypican 1, supporting a conserved role for glypican 1 in barrier function across species. In conclusion, we show that glypican 1 is a mechanosensor in the lung vasculature that mediates the effects of high pressure on barrier function by redox-sensitive pathways. This may be important for the progression of hydrostatic pulmonary edema in humans. Therapies targeting glypican 1 may be novel strategies to treat hydrostatic pulmonary edema.