Résumé
Background: The endothelial glycocalyx (EG) is a complex hair-like structure located on the apical membrane of endothelial cells, which is made up of proteoglycans (e.g. syndecan-1), glycosaminoglycans (e.g. hyaluronan), glycoproteins, and soluble plasma components. This structure plays critical roles in vascular physiology, including regulation of vascular permeability, mechanotransduction, and modulation of blood cells-endothelium interactions. Damage to the EG occurs in various pathologies including ischemia/reperfusion of several organs such as the kidney, atherosclerosis, and diabetes. EG damage leads to endothelial dysfunction and can be measured by the shedding of various components into the vascular stream. Therefore, rapid EG reconstruction in these conditions could be a highly valuable therapy.Aim: The aim of this master thesis was to study EG degradation in vitro and in vivo, in order to try, in the future, to promote its reconstruction.
Methods: EG degradation was studied in vitro using mouse glomerular endothelial cells, and in vivo after either heparinase or hyaluronidase injection in mice, or in a mouse model of left unilateral renal ischemia/reperfusion with right nephrectomy. Various methods were used to study EG degradation: transendothelial albumin passage measurement, confocal microscopy visualization of labelled EG components, enzyme-linked immunosorbent assays of shed components, and transmission electron microscopy.
Results: In vitro, several treatments (hyaluronidase from bovine testes, hyaluronidase from Streptomyces hyalurolyticus, lipopolysaccharides (LPS) and TNF-α) were able to degrade EG as confirmed by confocal microscopy. However, albumin passage across the endothelial barrier was not significantly increased, although LPS treatment (1 µg/mL) showed a trend towards increasing albumin permeability. These results were partially confirmed by investigation of EG shedding. Assay of EG degradation in mice treated with heparinase or hyaluronidase, or following renal ischemia/reperfusion, did not show signs of EG disruption as observed using confocal microscopy. However, syndecan-1 and hyaluronan shedding could be demonstrated in ischemic mice compared to control mice, while in comparison to sham-operated mice, only a trend could be shown.
Conclusion: In vitro, LPS treatment led to EG degradation and hyaluronan shedding, while in vivo, treatment of mice with heparinase or hyaluronidase did not seem to induce the expected EG degradation. However, EG disruption was demonstrated 24 h after renal ischemia/reperfusion in comparison to control mice. In the future, EG reconstruction using liposomal nanocarriers of preassembled glycocalyx or syndecan-1 could be considered after renal ischemia/reperfusion.
| la date de réponse | 18 janv. 2021 |
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| langue originale | Anglais |
| L'institution diplômante |
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| Superviseur | Bruno Flamion (Promoteur) & Sophie Dogne (Copromoteur) |