Sialic acids regulate microvessel permeability, revealed by novel in vivo studies of endothelial glycocalyx structure and function.

Kai B Betteridge,Rebecca R Foster,Christopher R Neal,Simon C Satchell,Andrew H J Salmon,Steven J Harper,Kenton P Arkill,David O Bates

doi:10.1113/jp274167

Abstract

Key points We have developed novel techniques for paired, direct, real‐time in vivo quantification of endothelial glycocalyx structure and associated microvessel permeability.Commonly used imaging and analysis techniques yield measurements of endothelial glycocalyx depth that vary by over an order of magnitude within the same vessel.The anatomical distance between maximal glycocalyx label and maximal endothelial cell plasma membrane label provides the most sensitive and reliable measure of endothelial glycocalyx depth.Sialic acid residues of the endothelial glycocalyx regulate glycocalyx structure and microvessel permeability to both water and albumin. The endothelial glycocalyx forms a continuous coat over the luminal surface of all vessels, and regulates multiple vascular functions. The contribution of individual components of the endothelial glycocalyx to one critical vascular function, microvascular permeability, remains unclear. We developed novel, real‐time, paired methodologies to study the contribution of sialic acids within the endothelial glycocalyx to the structural and functional permeability properties of the same microvessel in vivo. Single perfused rat mesenteric microvessels were perfused with fluorescent endothelial cell membrane and glycocalyx labels, and imaged with confocal microscopy. A broad range of glycocalyx depth measurements (0.17–3.02 μm) were obtained with different labels, imaging techniques and analysis methods. The distance between peak cell membrane and peak glycocalyx label provided the most reliable measure of endothelial glycocalyx anatomy, correlating with paired, numerically smaller values of endothelial glycocalyx depth (0.078 ± 0.016 μm) from electron micrographs of the same portion of the same vessel. Disruption of sialic acid residues within the endothelial glycocalyx using neuraminidase perfusion decreased endothelial glycocalyx depth and increased apparent solute permeability to albumin in the same vessels in a time‐dependent manner, with changes in all three true vessel wall permeability coefficients (hydraulic conductivity, reflection coefficient and diffusive solute permeability). These novel technologies expand the range of techniques that permit direct studies of the structure of the endothelial glycocalyx and dependent microvascular functions in vivo, and demonstrate that sialic acid residues within the endothelial glycocalyx are critical regulators of microvascular permeability to both water and albumin.

Highlights

The entire luminal surface of all vascular walls is covered with a continuous, carbohydrate-rich mesh: the endothelial glycocalyx (Weinbaum et al 2007)
The aim of this study was to understand the contribution of endothelial glycocalyx sialic acid residues to microvascular structure and permeability function, and to do so we have developed methods for real-time, direct, paired measurements of endothelial glycocalyx structure and function in vivo, as well as applying electron microscopy preservation to compare in vivo and ex vivo measurements of the same vessels with known functional properties
Estimates of endothelial glycocalyx depth made in vessels perfused with octadecyl rhodamine B chloride (R18) and fluorescein isothiocyanate (FITC)-wheat germ agglutinin (WGA) using the full-width half-maximum (FWHM) method yielded significantly higher measurements (1507 ± 136.9 nm, n = 10) than those determined in the same vessels using the peak to peak method of analysis (243.6 ± 22.44 nm, n = 10) (P < 0.001, paired t test; Fig. 3A)

Summary

Introduction

The entire luminal surface of all vascular walls is covered with a continuous, carbohydrate-rich mesh: the endothelial glycocalyx (Weinbaum et al 2007). The membrane-bound portion of the endothelial glycocalyx is supplemented with components of plasma to form a thickened endothelial surface layer This endothelial coat governs the nature of interactions between fluid, protein and cellular constituents of flowing blood and the endothelial cell, and is a critical regulator of vascular function (Curry & Adamson, 2013). Demonstrations of the resulting non-linear form of the Starling equation under steady-state filtration conditions (Michel & Phillips, 1987), and unequal actions of luminal and abluminal oncotic pressures (Hu et al 2000), support this theory These functional demonstrations are supported by recent observations that a repeating periodic arrangement of the fibres of the endothelial glycocalyx are sufficient to explain the near-uniform macromolecular permeability coefficients seen throughout the vasculature (Squire et al 2001; Arkill et al 2011). The endothelial glycocalyx regulates hydraulic resistance, with greater glycocalyx depths providing greater resistance (Adamson, 1990; Salmon et al 2009)

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