Composition of the endothelial glycocalyx and its relation to its thickness and diffusion of small solutes

Abstract

The endothelial glycocalyx is well endowed with the glycosaminoglycans (GAGs) heparan sulfate, chondroitin sulfate and hyaluronan. The current studies aimed to assess the relative contributions of each of these GAGs to the thickness and permeability of the glycocalyx layer by direct enzymatic removal of each using micropipettes to infuse heparinase, chondroitinase and hyaluronidase into post-capillary venules of the intestinal mesentery of the rat. The relative losses of GAGs due to enzymatic removal were compared with stimulated shedding of glycans induced by superfusing the mesentery with 10(-)(7)M fMLP. Thickness of the glycocalyx was assessed by infiltration of the glycocalyx with circulating FITC labeled 70kDa dextran (Dx70) and measuring the distance from the dye front to the surface of the endothelium (EC), which averaged 463nm under control conditions. Reductions in thickness were 43.3%, 34.1% and 26.1% following heparinase, chondroitinase and hyaluronidase, respectively, and 89.7% with a mixture of all three enzymes. Diffusion coefficients of FITC in the glycocalyx were determined using a 1-D diffusion model. By comparison of measured transients in radial intensity of a bolus of FITC with that of a computational model a diffusion coefficient D was obtained. Values of D were obtained corresponding to the thickness of the layer demarcated by Dx70 (D(Dx70)), and a smaller sublayer 173nm above the EC surface (D(173)), prior to and following enzyme infusion and superfusion with fMLP. The magnitude of D(Dx70) was twice that of D(173) suggesting that the glycocalyx is more compact near the EC surface. Chondroitinase and hyaluronidase significantly increased both D(Dx70) and D(173). However, heparinase decreased D(Dx70), and did not induce any significant change for the D(173). These observations suggest that the three GAGs are not evenly distributed throughout the glycocalyx and that they each contribute to permeability of the glycocalyx to a differing extent. The fMLP-induced shedding caused a reduction in glycocalyx thickness (which may increase permeability) and as with heparinase, decreased the diffusion coefficient of solutes (which may decrease permeability). This behavior suggests that the removal of heparan sulfate may cause a collapse of the glycocalyx which counters decreases in thickness by compacting the layer to maintain a constant resistance to filtration.

Fig. 1

Schematic of the experiment protocol. A post-capillary venule in the exteriorized rat mesentery was selected for intravital microscopic observation. An upstream branch was cannulated with a micropipette for perfusion with Alexa labeled BS-1 lectin to label the glycocalyx, or enzymes for GAG degradation. Fluorescence intensity of the endothelial surface was acquired to quantify glycan shedding.

Fig. 2

Fluorescent labeling of the endothelial cell (EC) glycocalyx. (A) Brightfield image of a post-capillary venule (diameter = 40.7 μm). The plasma membrane of the EC was taken as the outermost edge of the dark refractive band between the EC and plasma layer. (B) Fluorescence image 10-min following proximal micropipette infusion of BS1-Alexa lectin. In this example fluorescence was confined to the left microvessel wall due to heterogeneity of network perfusion. A measurement line was drawn along the left EC wall and fluorescence intensity was averaged over an area within 0.5 μm on either side of the measurement line.

Fig. 3

Measurement of glycocalyx thickness. (A) Brightfield image of a post-capillary venule (dia = 35 μm) after a 0.15 mL systemic FITC-Dx70 bolus injection. The outermost edge of the dark refractive band was taken as the surface of the EC. (B) Fluorescence micrograph with circulating FITC-Dx70. (C) Fluorescence intensity distribution (open symbols) along a radial measurement line was fit with a sigmoidal curve (solid line) to determine its inflection point, IP. The distance between the IP and the EC surface was taken as the thickness of the glycocalyx.

Fig. 4

Fluorescence intensity of BS1-Alexa along the endothelial surface of post-capillary venules 30-40 min following proximal infusion of the lectin with a micropipette. Control measurements were taken 10 min prior to each treatment. Intensities were normalized with respect to control, I_Treated/I_Control. Intensity of the fluorescent stain fell 15% with no stimulus, due to natural shedding of glycans. Following 10-min of enzymatic degradation with heparinase, chondroitinase and hyaluronidase, and superfusion of the mesentery with fMLP, glycan labeling was reduced significantly compared to natural shedding (no stimulus), *p < 0.05.

Fig. 5

Estimation of the thickness of the glycocalyx from the thickness of the barrier to infiltration of FTIC-Dx70. (A) Thickness measurements taken as the distance between the inflection point in the radial intensity profile at the wall and the EC surface, for control (no perfusion) and micropipette perfusion with the indicated enzymes, and superfusion with fMLP. (B) Ratio of the post to pre-treatment thickness, δ_Treated/δ_Control. The number of observations is given along with the number of post-capillary venules (in parenthesis). All treatments caused a significant decrease (*p<0.05) relative to control measurements.

Fig. 6

Radial concentration at the wall of a post-capillary venule following systemic infusion (jugular vein, i.v.) of a small solute (FITC). Measured fluorescence intensity profiles (○) were obtained with time, normal to venular wall. The shaded surface represents the solution to the 1-D diffusion model, computed using the measured intensity-time curve at a distance δ from the wall, determined by the exclusion of FITC-Dx70 (Fig. 5). In this illustrative case, the experimental data and the computational prediction agreed within an RMS error of 34.6%, and correspond to a diffusion coefficient for FITC of 2.61×10⁻⁹ cm²/sec.

Fig. 7

Calculated diffusion coefficient, D, of FITC in the glycocalyx obtained from a model of unsteady one dimensional diffusion normal to the EC surface. (A) Diffusion coefficient (D_Dx70) from solution of the diffusion equation based upon time variation of FITC concentration at a distance from the EC surface equal to the exclusion thickness of Dx70. (B) Diffusion coefficient (D₁₇₃) assessed for a sublayer 173 nm above the EC surface. Neither heparinase nor fMLP significantly affected D₁₇₃. The number of observations is given along with the number of post-capillary venules in parentheses. *Significantly different from control, p<0.05.