Three-dimensional ultrastructure of capillary endothelial glycocalyx under normal and experimental endotoxemic conditions

Abstract

Background: Sugar-protein glycocalyx coats healthy endothelium, but its ultrastructure is not well described. Our aim was to determine the three-dimensional ultrastructure of capillary endothelial glycocalyx in the heart, kidney, and liver, where capillaries are, respectively, continuous, fenestrated, and sinusoidal.

Methods: Tissue samples were processed with lanthanum-containing alkaline fixative, which preserves the structure of glycocalyx.

Results: Scanning and transmission electron microscopy revealed that the endothelial glycocalyx layer in continuous and fenestrated capillaries was substantially thicker than in sinusoids. In the heart, the endothelial glycocalyx presented as moss- or broccoli-like and covered the entire luminal endothelial cell surface. In the kidney, the glycocalyx appeared to nearly occlude the endothelial pores of the fenestrated capillaries and was also present on the surface of the renal podocytes. In sinusoids of the liver, glycocalyx covered not only the luminal side but also the opposite side, facing the space of Disse. In a mouse lipopolysaccharide-induced experimental endotoxemia model, the capillary endothelial glycocalyx was severely disrupted; that is, it appeared to be peeling off the cells and clumping. Serum concentrations of syndecan-1, a marker of glycocalyx damage, were significantly increased 24 h after administration of lipopolysaccharide.

Conclusions: In the present study, we visualized the three-dimensional ultrastructure of endothelial glycocalyx in healthy continuous, fenestrated, and sinusoidal capillaries, and we also showed their disruption under experimental endotoxemic conditions. The latter may provide a morphological basis for the microvascular endothelial dysfunction associated with septic injury to organs.

Keywords: Capillary; Endothelial cell; Glycocalyx; Sepsis; Ultrastructure; Vascular endothelial injury.

Fig. 1

Scanning and transmission electron microscopy showing glycocalyx of continuous capillaries in the heart under normal conditions. a1 Cardiac capillary without lanthanum nitrate staining. a2 Expanded view of the area within the red rectangle square in (a1). Continuous capillaries in the heart have a continuous thin basement membrane. b1 Cardiac capillary with lanthanum nitrate staining. The endothelial glycocalyx, which is the bush-like structure, can be seen on the surface of the vascular endothelium. b2 Backscattered electron microscopic image of the same specimen as in (b1). The location of backscattered electrons is consistent with the bush-like structure. c1 Energy-dispersive spectroscopic image of a cardiac capillary stained with lanthanum nitrate. c2 Ingredient analysis of the area within the red rectangle in (c1). The bush-like structure includes lanthanum, indicating this structure is the endothelial glycocalyx. C Carbon, O Oxygen, P Phosphorus, S Silicon, La Lanthanum. d1 Transmission electron microscopic imaging of cardiac capillary with lanthanum nitrate staining. d2 Expanded view of the area within the red rectangle in (d1). The endothelial glycocalyx can also be seen on the surface of the vascular endothelium

Fig. 2

Scanning electron microscopy showing glycocalyx in fenestrated capillaries of the kidney and sinusoids of the liver under normal conditions. a Ultrastructure of glomerular capillaries under normal conditions. a1 Fenestrated capillary without lanthanum nitrate staining. Small pores are present on the surface of the endothelial cells. a2, a3 Lanthanum nitrate staining to visualize endothelial glycocalyx. a3 Expanded view of the area within the red rectangle in (a2). Endothelial glycocalyx covers the surface of glomerular capillaries. b Ultrastructure of podocytes on the outer surface of the glomerulus under normal condition. b1 Podocytes without lanthanum nitrate staining. Many podocytes firmly intertwine with each other to form a meshwork. b2, b3 Glycocalyx on podocytes visualized by lanthanum nitrate staining. b3 Expanded view of the area within the red rectangle in (b2). Glycocalyx overlays the surface of podocytes. c Ultrastructure of hepatic sinusoids under normal conditions. c1 Sinusoid without lanthanum nitrate staining. Sinusoids in liver are open-pore capillaries. c2, c3 Visualized glycocalyx in sinusoids. c3 Expanded view of the area within the red rectangle in (c2). The endothelial glycocalyx in sinusoids does not overlay the open fenestrations but is also present in the space of Disse

Fig. 3

Transmission electron microscopy showing glycocalyx in glomerular fenestrated capillaries and hepatic sinusoids under normal conditions. a Ultrastructure of glomerular capillaries under normal conditions. a1 Glomerular capillary without lanthanum nitrate staining. The healthy glomerular endothelium is composed of three layers, including endothelial cells (black arrow), as well as basement membrane and podocytes (red arrow), which are bound with each other. a2, a3 Lanthanum nitrate staining to visualize glycocalyx. a3 Expanded view of the area within the red rectangle in (a2). Glycocalyx is present on the surface of glomerular capillaries and podocytes. b Ultrastructure of hepatic sinusoids under normal conditions. b1 Sinusoid without lanthanum nitrate staining. The sinusoid is composed of discontinuous flat endothelial cells (black arrow) and has large pores. The space of Disse is situated under the endothelium (red arrow). b2, b3 Visualized glycocalyx in sinusoids. b3 Expanded view of the area within the red rectangle in (b2). The endothelial glycocalyx layer of sinusoids is thin (black arrow) and is also present in the space of Disse (red arrow)

Fig. 4

Scanning electron microscopy showing glycocalyx in continuous, fenestrated, and sinusoidal capillaries under septic conditions. a Ultrastructure of continuous capillaries in the heart under septic conditions. a1 Continuous capillary without lanthanum nitrate staining. Thickening of the endothelial wall is presumed to be due to edematous changes related to inflammation. a2, a3 Lanthanum nitrate staining to visualize the endothelial glycocalyx. a3 Expanded view of the area within the red rectangle in (a2). The endothelial glycocalyx is peeled away from the surface of endothelial cells, and the residue is found inside the vascular lumen (white arrow). b Ultrastructure of glomerular capillaries under septic conditions. b1 Fenestrated capillary without lanthanum nitrate staining. Destruction of the small pore structure is observable. In addition, the endothelial wall appears edematous. b2, b3 Lanthanum nitrate staining to visualize the endothelial glycocalyx. b3 Expanded view of the area within the red rectangle in (b2). Glycocalyx is cast off from the endothelial cells, and the residue of it exists inside the vascular lumen (white arrow). c Ultrastructure of hepatic sinusoids under septic conditions. c1 Sinusoid without lanthanum nitrate staining. The large pores are nearly completely occluded (white arrow). c2, c3 Visualized glycocalyx within sinusoids. c3 Expanded view of the area within the red rectangle in (c2). The sinusoidal endothelial glycocalyx is peeled away from the endothelial cells, and the residue is present inside the vascular lumen (white arrow)

Fig. 5

Transmission electron microscopy showing glycocalyx in capillaries under septic conditions. a Ultrastructure of cardiac capillaries under septic conditions. a1 Continuous capillary without lanthanum nitrate staining. The capillary wall appears edematous, and there is fibrin deposited inside the capillary lumen. a2, a3 Lanthanum nitrate staining to visualize the endothelial glycocalyx. a3 Expanded view of the area within the red rectangle in (a2). The endothelial glycocalyx is peeled away, and there is little glycocalyx on the endothelial cells (red arrow). b Ultrastructure of glomerular capillaries under septic conditions. b1 Glomerular capillary without lanthanum nitrate staining. There is a gap between the podocytes and basement membrane under septic conditions (red arrows). b2, b3 Lanthanum nitrate staining to visualize the glycocalyx. b3 Expanded view of the area within the red rectangle in (b2). The glycocalyx is cast off from the surface of the glomerular endothelial cells and podocytes. c Ultrastructure of hepatic sinusoids under septic conditions. c1 Sinusoid without lanthanum nitrate staining. Whereas the sinusoid is normally composed of discontinuous flat endothelial cells, here the endothelial cells have become edematous, and the large pores are closed (red arrow). c2, c3 Visualized glycocalyx in sinusoids. c3 Expanded view of the area within the red rectangle in (c2). The endothelial glycocalyx layer of sinusoids has peeled off, and the space of Disse has become unclear