Imaging the endothelial glycocalyx in vitro by rapid freezing/freeze substitution transmission electron microscopy

Abstract

Objective: Recent publications questioned the validity of endothelial cell (EC) culture studies of glycocalyx (GCX) function because of findings that GCX in vitro may be substantially thinner than GCX in vivo. The assessment of thickness differences is complicated by GCX collapse during dehydration for traditional electron microscopy. We measured in vitro GCX thickness using rapid freezing/freeze substitution (RF/FS) transmission electron microscopy (TEM), taking advantage of the high spatial resolution provided by TEM and the capability to stably preserve the GCX in its hydrated configuration by RF/FS.

Methods and results: Bovine aortic EC (BAEC) and rat fat pad EC were subjected to conventional or RF/FS-TEM. Conventionally preserved BAEC GCX was ≈0.040 μm in thickness. RF/FS-TEM revealed impressively thick BAEC GCX of ≈11 μm and rat fat pad EC GCX of ≈5 μm. RF/FS-TEM also discerned GCX structure and thickness variations due to heparinase III enzyme treatment and extracellular protein removal, respectively. Immunoconfocal studies confirmed that the in vitro GCX is several micrometers thick and is composed of extensive and well-integrated heparan sulfate, hyaluronic acid, and protein layers.

Conclusions: New observations by RF/FS-TEM reveal substantial GCX layers on cultured EC, supporting their continued use for fundamental studies of GCX and its function in the vasculature.

Figure 1

TEM of GCX-covered BAEC (A) preserved conventionally, labeled with ruthenium red and osmium tetroxide, and alcohol dehydrated, or (B) preserved by RF/FS and osmium tetroxide stained (stars (*) denote abluminal GCX). (C) High magnification image of conventionally preserved BAEC GCX (arrowheads (▼) denote extended strands of GCX). (D) High magnification image of RF/FS preserved BAEC GCX, showing (from left to right) locations near the cell membrane, further away from the cell membrane, in the center region of the GCX, and at the most apical surface of the GCX .

Figure 2

RF/FS preservation, osmium tetroxide staining, and TEM of (A) untreated BAEC GCX, (B) untreated RFPEC GCX, (C) BAEC GCX treated with Heparanase III to degrade the heparan sulfate component of the GCX, and (D) GCX of BAEC cultured in the absence of FBS and BSA.

Figure 3

The GCX thickness measurements, differences between thicknesses of various cell types and/or treatments, standard errors, and statistical analysis demonstrate the validity and stability of the RF/FS TEM approach. BAEC GCX thickness was 11.35 ± 0.21 μm (n = 7 sections) with RF/FS-TEM, but 0.042 ± 0.003 μm (n = 3 sections) with conventional-TEM. 5.83 ± 1.13 μm (n = 3 sections) RFPEC GCX thickness was determined by RF/FS-TEM. BAEC GCX was 11.98 ± 0.73 μm (n = 4 sections) with heparinase III treatment and undetectable without protein. *P < 0.05.

Figure 4

Confocal micrographs of EC HS (red), HA (green), BSA (green), and nucleii (blue). (A) BAEC HS, (B) BAEC HA, (C) BAEC BSA, (D) RFPEC HS, (E) RFPEC HA, and (F) RFPEC BSA are shown. Bar = 5 um. (G) Average ± SEM GCX thickness (n = GCX-covered EC nucleii), maximum GCX thickness per cross-section, and junctional GCX thickness. Arrowheads: locations of cell-to-cell junctions.

Figure 5

This cartoon, redrawn from Pahakis et al. on the basis of Heuser’s review, depicts how live conditions, aldehyde fixation, and aldehyde combined with dehydration differentially affect GCX thickness and ultrastructural composition.