Abstract
The endothelial glycocalyx (eGC), a carbohydrate-rich layer lining the luminal side of the endothelium, regulates vascular adhesiveness and permeability. Although central to the pathophysiology of vascular barrier dysfunction in sepsis, glycocalyx damage has been generally understudied, in part because of the aberrancy of in vitro preparations and its degradation during tissue handling. The aim of this study was to analyze inflammation-induced damage of the eGC on living endothelial cells by atomic-force microscopy (AFM) nanoindentation technique. AFM revealed the existence of a mature eGC on the luminal endothelial surface of freshly isolated rodent aorta preparations ex vivo, as well as on cultured human pulmonary microvascular endothelial cells (HPMEC) in vitro. AFM detected a marked reduction in glycocalyx thickness (266 ± 12 vs. 137 ± 17 nm, P<0.0001) and stiffness (0.34 ± 0.03 vs. 0.21 ± 0.01 pN/mn, P<0.0001) in septic mice (1 mg E. coli lipopolysaccharides (LPS)/kg BW i.p.) compared to controls. Corresponding in vitro experiments revealed that sepsis-associated mediators, such as thrombin, LPS or Tumor Necrosis Factor-α alone were sufficient to rapidly decrease eGC thickness (-50%, all P<0.0001) and stiffness (-20% P<0.0001) on HPMEC. In summary, AFM nanoindentation is a promising novel approach to uncover mechanisms involved in deterioration and refurbishment of the eGC in sepsis.
Highlights
Endothelial hyperpermeability is a hallmark of systemic inflammatory response syndrome (SIRS) and sepsis that largely contributes to high morbidity and mortality in critically-ill patients
The first slope represents the stiffness of the endothelial glycocalyx (eGC), whereas the second steep slope reflects the stiffness of the plasma membrane with the underlying cortical actin web (Figure 2 B)
All of the above mentioned results were highly reproducible. This data indicates that enzymatic removal of any eGC constituents alters its nanomechanics in a relatively rapid and uniform fashion
Summary
Endothelial hyperpermeability is a hallmark of systemic inflammatory response syndrome (SIRS) and sepsis that largely contributes to high morbidity and mortality in critically-ill patients. Inflammation-induced vascular leakage has long been ascribed to a malfunction of the endothelial cell itself. Recent studies provided compelling evidence that the endothelium is protected against pathogenic insults by a highly hydrated negatively charged “firewall” on the luminal side called the glycocalyx [4,5,6]. Given its strategic location as the interface between the blood and the endothelium, the intact glycocalyx mediates flow-induced shear stress on endothelial cells, prevents transvascular protein leakage and reduces leukocyteendothelial interactions [5,7,8]. The endothelial glycocalyx (eGC) is a carbohydrate-rich gellike mesh of large anionic polymers covering the luminal surface of endothelium along the entire vascular tree. The structure of the glycocalyx layer is fairly stable but subject to a permanent dynamic balance between biosynthesis of new glycosaminoglycans and shear dependent removal of existing constituents
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