Time-Dependent Dynamics Required for the Degradation and Restoration of the Vascular Endothelial Glycocalyx Layer in Lipopolysaccharide-Treated Septic Mice

The endothelial glycocalyx (GCX) plays a key role in the development of organ failure following sepsis. Researchers have investigated GCX degradation caused by pathological conditions. Nonetheless, the GCX restoration process remains poorly understood. Herein, we developed a model in which GCX restoration could be reproduced in mice using in vivo imaging and a dorsal skinfold chamber (DSC). The severity of sepsis was controlled by adjusting the dose of lipopolysaccharide (LPS) used to trigger GCX degradation in BALB/c mice. We evaluated the GCX thickness, leukocyte-endothelial interactions, and vascular permeability using in vivo imaging through DSC under intravital microscopy. The plasma concentration of syndecan-1(Sdc-1), a GCX structural component, was also determined as a marker of GCX degradation. Thus, we developed a reproducible spontaneous GCX recovery model in mice. Degraded GCX was restored within 24 h by the direct visualization of the endothelial GCX thickness, and leukocyte-endothelial interactions. In contrast, indirectly related indicators of recovery from sepsis, such as body weight and blood pressure, required a longer recovery time. This model can be used to study intractable angiopathy following sepsis.

Changes in endothelial glycocalyx layer protective ability after inflammatory stimulus

Leukocyte adhesion to the endothelium is an important early step in the initiation and progression of sepsis. The endothelial glycocalyx layer (EGL) has been implicated in neutrophil adhesion and barrier dysfunction, but studies in this area are few. In this report we examine the hypothesis that damage to the structure of the EGL caused by inflammation leads to increased leukocyte adhesion and endothelial barrier dysfunction. We used human umbilical vein endothelial cells (HUVECs) enzymatically treated to remove the EGL components hyaluronic acid (HA) and heparan sulfate (HS) as a model for EGL damage. Using atomic force microscopy, we show reductions in EGL thickness after removal of either HA or HS individually, but the largest decrease, comparable to TNF-a treatment, was observed when both HA and HS were removed. Interestingly, removal of HS or HA individually did not affect neutrophil adhesion significantly, but removal of both constituents resulted in increased neutrophil adhesion. To test EGL contributions to endothelial barrier properties, we measured trans-endothelial electrical resistance (TEER) and diffusion of fluorescently labeled dextran (10 kDa MW) across the monolayer. Removal of EGL components decreased TEER, but had an insignificant effect on dextran diffusion rates. The reduction in TEER suggests that disruption of the EGL may predispose endothelial cells to increased rates of fluid leakage. These data support the view that damage to the EGL during inflammation has significant effects on the accessibility of adhesion molecules, likely facilitates leukocyte adhesion, and may also contribute to increased rates of fluid transport into tissues.

Microvascular ion transport through endothelial glycocalyx layer: new mechanism and improved Starling principle

Ion transport through the endothelial glycocalyx layer is closely associated with many vascular diseases. Clarification of ion behaviors around the endothelial glycocalyx layer under varying circumstances will benefit pathologies related to cardiovascular and renal diseases. In this research, a series of large-scale molecular dynamics simulations are conducted to study the response of ion transport to the changing blood flow velocity and the shedding of endothelial glycocalyx sugar chains. Results indicate that blood flow promotes the outward Na+ transport from the near-membrane region to the lumen via the endothelial glycocalyx layer. Scrutiny of sugar-chain dynamics and their interactions with Na+ suggests that corner conformation of endothelial glycocalyx sugar chains confines the movement of the Na+, whereas stretching conformation facilitates the motion of Na+ ions. The flow impact on ion transport of Na+ is nonlinear. Based on the findings, the Starling principle and its revised version, which are prevailingly used to predict the ion transport of the endothelial glycocalyx layer, are further improved. An estimation based on the further revised Starling principle indicates that physiological flow changes the osmotic part of transendothelial water flux by 8% compared with the stationary situation. NEW & NOTEWORTHY The biophysical roles of negatively charged oligosaccharides of the endothelial glycocalyx have gained increasing attention due to their importance in regulating microvascular fluid exchange. The Starling principle and its revisions are at the heart of the understanding of fluid homeostasis in the periphery. Here, the blood flow changes the conformations of glycocalyx sugar chains, thereby influencing availability of Na+ for transport. Based on the findings, the Starling principle and its revision are further improved.