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. 2018 Aug 12;19(8):2375.
doi: 10.3390/ijms19082375.

Glycocalyx Preservation and NO Production in Fatty Livers-The Protective Role of High Molecular Polyethylene Glycol in Cold Ischemia Injury

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Free PMC article

Glycocalyx Preservation and NO Production in Fatty Livers-The Protective Role of High Molecular Polyethylene Glycol in Cold Ischemia Injury

Alexandre Lopez et al. Int J Mol Sci. .
Free PMC article

Abstract

Improving the protection of marginal liver grafts during static cold storage is a major hurdle to increase the donor pool of organs. The endothelium glycocalyx quality of preservation influences future inflammatory and oxidative responses. One cellular pathway responsible for the formation of nitric oxide by endothelial cells is dependent on the stimulation of proteoglycans present in the glycocalyx. We investigated the impact of the glycocalyx preservation in static cold storage of fatty liver preserved in different preservation solutions on the endothelium-mediated production of NO. Zucker fatty rat livers were preserved 24 h in static cold storage in either Institut Georges Lopez-1 (IGL-1) (n = 10), IGL-0 (i.e., without PEG35) (n = 5) or Histidine-Tryptophan-Ketoglutarate (HTK) (n = 10) preservation solutions before being processed for analysis. For Sham group (n = 5), the fatty livers were immediately analyzed after procurement. The level of transaminases and nitrites/nitrates were measured in the washing perfusate. Glycocalyx proteins expressions, Syndecan-1, glypican-1 and heparan sulfate (HS), were determined in the tissue (ELISA). Steatotic livers preserved 24 h in IGL-1 preservation solution have a significant lower level of transaminases (aspartate aminotransferase (AST), alanine aminotransferase (ALT)) and less histological damages than steatotic livers preserved 24 h with HTK (p = 0.0152). The syndecan-1 is significantly better preserved in IGL-1 group compared to HTK (p < 0.0001) and we observed the same tendency compared to IGL-0. No significant differences were observed with glypican-1. HS expression in HTK group was significantly higher compared to the three other groups. HS level in IGL-1 was even lower than IGL-0 (p = 0.0005) which was similar to Sham group. The better protection of the glycocalyx proteins in IGL-1 group was correlated with a higher production of NO than HTK (p = 0.0055) or IGL-0 (p = 0.0433). IGL-1 protective mechanisms through the formation of NO could be due to its better protective effects on the glycocalyx during SCS compared to other preservation solutions. This beneficial effect could involve the preservation state of syndecan-1 and the internalization of HS.

Keywords: cold storage; glycocalyx; ischemia; liver; polyethylene glycol; steatosis.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Fatty liver ischemia injury. Transaminases were collected after procurement (Sham group) or after 24 h of cold preservation in IGL-1, HTK or IGL-0 solutions (IGL-1, HTK and IGL-0 group, respectively). Transaminases ALT (a) and AST (b) release were higher in the HTK group compared to the three groups and IGL-0 transaminases were higher than Sham and IGL-1 groups. Results are presented as mean (SD), IGL-1/HTK n = 10/group, Sham/IGL-0 n = 5/group, * p < 0.05 vs. Sham; # p < 0.05 vs. IGL-1 (One-way ANOVA).
Figure 2
Figure 2
Fatty livers histology after cold ischemia. (a) Tissue samples were stained with HES (×10). Using Sham group (A) as a reference, IGL-1 group (B) showed less histological injury (i.e., cell dissociation, loss of hepatic architecture, swollen hepatocytes) than HTK (C) and IGL-0 (D) groups. (b) Damage grade score was made according to the preservation state of the samples evaluated by a pathologist (E).* p < 0.05 vs. Sham; # p < 0.05 vs. IGL-1 (Kruskal–Wallis test).
Figure 3
Figure 3
Glycocalyx protection in fatty livers after 24 h of cold storage. Protein expression of the three main components of the glycocalyx were measured by ELISA from tissue samples. (a) Syndecan-1 level was lower in HTK group compared to Sham and IGL-1 groups; (b) glypican-1 expression was the same in all the groups; (c) Heparan sulfate expression was lower in IGL-1 group compared to the three other groups and the opposite for HTK which expressed a higher level of HS. Results are presented as mean (SD), IGL-1/HTK n = 10/group, Sham/IGL-0 n = 5/group, * p < 0.05 vs. Sham; # p < 0.05 vs. IGL-1; ° p < 0.05 vs. HTK (One-way ANOVA).
Figure 4
Figure 4
Nitrites/Nitrates expression after 24 h cold ischemia in steatotic livers. NO2/NO3 concentration was measured using a kit. HTK group production of NO2/NO3 was greatly reduced compared to Sham and IGL-1. The same decrease in the production of NO was observed when the PEG35 was removed (IGL-0) compared to IGL-1. NO2/NO3 in fatty liver preserved 24 h in IGL-0 was almost double compared to the Sham group. Results are presented as mean (SD), IGL-1/HTK n = 10/group, Sham/IGL-0 n = 5/group, * p < 0.05 vs. Sham; # p < 0.05 vs. IGL-1 (One-way ANOVA).
Figure 5
Figure 5
Endothelial glycocalyx-mediated NO production. (a) Internalization of the glypican-1 in caveolae is possible thanks to the stabilization of the actin filament by the transmembrane syndecan-1. The heparan sulfate is then cleaved to further increase the production of NO in the cells. This glycocalyx-mediated NO induces a vasodilatation of the endothelium and improves the steatotic graft condition; (b) in a graft with higher cold ischemia injury, the lower quantity of syndecan-1 is not sufficient to stabilize the cytoskeleton, which inhibits the formation of caveolae, leading to the absence of NO production.

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