Fluorescence correlation spectroscopy can probe albumin dynamics inside lung endothelial glycocalyx

Abstract

The endothelial glycocalyx is believed to play a major role in capillary permeability by functioning as a macromolecular barrier overlying the intercellular junction. Little is known about the functional attributes of the glycocalyx (i.e., porosity and permeability) or which constituents contribute to its overall structure-function relationship. In this report, we demonstrate the utility of fluorescence correlation spectroscopy (FCS) to measure albumin diffusion rates and concentration profiles above the cell surface and overlying the intercellular junctions of lung capillary endothelial cells. Albumin diffusion rates and concentration profiles were obtained before and after enzymatic digestion of the glycocalyx with pronase, heparanase, or hyaluronidase. The results suggest a structure interacting with albumin located from 1.0 to 2.0 microm above the cell membrane capable of reducing albumin diffusion by 30% while simultaneously increasing albumin concentration fivefold. Digestion of the glycocalyx with pronase or heparanase resulted in only modest changes in albumin diffusion and concentration profiles. Hyaluronidase digestion completely eliminated albumin-glycocalyx interactions. These data also suggest that hyaluronan is a major determinant for albumin interactions with the lung endothelial glycocalyx. Confocal images of heparan sulfate and hyaluronan confirm a cell-surface layer 2-3 mum in thickness, thus supporting FCS measurements. In summary, we report the first use of FCS to probe extracellular structures and further our understanding of the structure-function relationship of the lung microvascular endothelial glycocalyx.

Effect of Surface Charge of Immortalized Mouse Cerebral Endothelial Cell Monolayer on Transport of Charged Solutes

“…The method for permeability measurement on rat pial microvessels has been published in our previous studies. 39,40 The permeability of bEnd3 monolayer and that of rat pial microvessels to QAbCD nanoparticles are summarized in Expt. data (ORO) FIGURE 7.…”

“…There is also ample evidence from other studies that a substantial SGL exists in vitro depending on the culture conditions. 6,[40][41][42] Under our culture conditions for bEnd3 cells, we tested the SGL as a functional transport barrier. To confirm its structural properties, we will perform immunostaining to relevant SGL components and confocal microscopy in the future.…”

The endothelial glycocalyx: a review of the vascular barrier

“…Even very low concentrations of albumin help maintain endothelial barrier function [51]. Albumin attenuated glycocalyx shedding during 4 h of cold ischaemia and reperfusion in guinea pig hearts, reducing interstitial oedema and intracoronary adhesion of leucocytes [40].…”

Novel regulators of endothelial barrier function

“…Based on studies from vascular beds of systemic vessels, the glycocalyx has been shown to act as a sieve allowing transendothelial transport of low-molecularweight solutes, inhibiting red blood cell and neutrophil adhesion and extravasation, and as a mechanotransducer (67,113). However, characterization of the glycocalyx in the pulmonary microcirculation remained controversial as most of the studies relied on using enzymatic degradation of glycocalyx constituents (24, 31, 35, 37, 45,71,102,130). Recently, Schmidt and coworkers (125) used a model of intravital microscopy in the intact, ventilated mouse (136) to demonstrate that the pulmonary endothelial glycocalyx is ϳ1.7 m thick, i.e., around two to three times the value of the glycocalyx in the systemic circulation.…”