Abstract 450: Tumor Necrosis Factor Disrupts Endothelial Surface Glycocalyx Imaged by High-pressure Freezing, Freeze Substitution TEM
Objective: The endothelial surface glycocalyx (GCX) is a hydrated mesh formed from a wide variety of endothelial cell membrane-associated macromolecules including glycoproteins bearing sialic acid and proteoglycans. It is thought to be damaged in diseases associated with elevated levels of tumor necrosis factor α (TNF-α) since surface proteoglycan and sialic acid amount decreased after TNF treatment. We imaged in vitro GCX in response to TNF-α using rapid freezing/freeze substitution (RF/FS) transmission electron microscopy (TEM) so that the GCX can be stably preserved in its hydrated configuration.
Methods and Results: Human glomerular ECs (HGenC) were seeded onto fibronectin- coated PET membrane of the 96-multiwell inserts and allowed to grow to confluence for 4 days. Before experimentation, cells were then treated with TNF (20 ng/ml) or control medium for 24 h. The PET membrane with the cells attached were removed from the Transwell insert by carefully cutting around the membrane edges with a scalpel, and transferred to aluminum planchettes (Ted Pella). The membranes were then subjected to RF/FS-TEM. RF/FS-TEM revealed impressively thick HGenC GCX of ≈13 μm under control condition. In cells treated with TNF, the GCX completely disappeared. Immunoconfocal studies confirmed that in vitro GCX was damaged by TNF.
Conclusions: New observations by RF/FS-TEM reveal substantial GCX layers on cultured human glomerular EC, which are completely abolished by TNF-α.
Author Disclosures: C. Xu: None. J. Austin: None. B. Hack: None. P. Cunningham: None.
- © 2015 by American Heart Association, Inc.