Sulodexide improves vascular permeability via glycocalyx remodelling in endothelial cells during sepsis

Abstract

Background: Degradation of the endothelial glycocalyx is critical for sepsis-associated lung injury and pulmonary vascular permeability. We investigated whether sulodexide, a precursor for the synthesis of glycosaminoglycans, plays a biological role in glycocalyx remodeling and improves endothelial barrier dysfunction in sepsis.

Methods: The number of children with septic shock that were admitted to the PICU at Children's Hospital of Fudan University who enrolled in the study was 28. On days one and three after enrollment, venous blood samples were collected, and heparan sulfate, and syndecan-1 (SDC1) were assayed in the plasma. We established a cell model of glycocalyx shedding by heparinase III and induced sepsis in a mouse model via lipopolysaccharide (LPS) injection and cecal ligation and puncture (CLP). Sulodexide was administrated to prevent endothelial glycocalyx damage. Endothelial barrier function and expression of endothelial-related proteins were determined using permeability, western blot and immunofluorescent staining. The survival rate, histopathology evaluation of lungs and wet-to-dry lung weight ratio were also evaluated.

Results: We found that circulating SDC1 levels were persistently upregulated in the non-alive group on days 1 and 3 and were positively correlated with IL-6 levels. Receiver operating characteristic curve analysis showed that SDC1 could distinguish patients with mortality. We showed that SDC1-shedding caused endothelial permeability in the presence of heparinase III and sepsis conditions. Mechanistically, sulodexide (30 LSU/mL) administration markedly inhibited SDC1 shedding and prevented endothelial permeability with zonula occludens-1 (ZO-1) upregulation via NF-κB/ZO-1 pathway. In mice with LPS and CLP-induced sepsis, sulodexide (40 mg/kg) administration decreased the plasma levels of SDC1 and increased survival rate. Additionally, sulodexide alleviated lung injury and restored endothelial glycocalyx damage.

Conlusions: In conclusion, our data suggest that SDC1 predicts prognosis in children with septic shock and sulodexide may have therapeutic potential for the treatment of sepsis-associated endothelial dysfunction.

Keywords: Syndecan-1; endothelium barrier function; glycocalyx remodeling; sepsis; sulodexide.

Figure 1

Syndecan-1 (SDC1) is a prognosis biomarker of children with septic shock. (A) ELISA to detect the levels of SDC1and IL-6 in plasma from 28 children with sepsis within 24 hours of PICU admission (day 1). (B) Correlation between levels of IL-6 and SDC1. (C) Changes of levels of SDC1 and heparan sulfate in plasma from day 1 to day 3. (D) ROC curve analysis of SDC1 expression on day 1 between survival group and non-survival group. Bars and error bars represent the mean ± SEM; *p < 0.05, **p < 0.01.

Figure 2

Sulodexide decreased heparinase III-induced shedding of SDC1 in MLMECs. (A) MLMECs were treated with 15 mU/mL Hep III for 2 h, 4 h, or 8 h. Cells in Hep III+SDX group were treated with 30 LSU/mL sulodexide for 2 h before. Cells in the SDX group were treated with sulodexide for 2 h, and then with PBS in the same volume. Representative image of immunofluorescence of SDC1 on MLMECs, scale bar = 200 μm. (B) Densitometry of immunofluorescence of SDC1 on MLMECs. (C) ELISA to detect the levels of SDC1 in MLMECs supernatant with treatment of Hep III for 2 h. Data were expressed as the means ± SEM; *p < 0.05.

Figure 3

Sulodexide improved endothelial permeability resulting from glycocalyx shedding-induced ZO-1 disruption. (A) Pattern diagram for transwell model. MLMECs were treated with 15 mU/mL Hep III for 2 h, 4 h or 8 h, and/or 30 LSU/mL SDX for 2 h, respectively. (B) Relative fluorescence value of FD40 that passed through the inserts was assayed for 2 h, 4 h, or 8 h. (C) Levels of ZO-1 and VE-cadherin were quantified by western blot for 2 h or 4 h. (D) Statistical analysis of the levels of ZO-1. Data were expressed as means ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4

Sulodexide promoted permeability via abolishing activation of NF-κB signaling (A, B) MLMECs (A)/HUVECs (B) were treated with 15 mU/mL Hep III for 15/30 min. Cells in the Hep III+SDX group were pre-treated with 30 LSU/mL sulodexide for 2 h. Cells in the SDX group were treated with sulodexide for 2 h without Hep III. (C, D) MLMECs (C)/HUVECs (D) were treated with 40 μM/mL Bay 11- 7082 for 12 h. Cells were then treated with or without 15 mU/mL Hep III for 15/30 min. The levels of NF-κB/p-p65 and NF-κB/p65 were quantified by western blot. (E, F) MLMECs €/HUVECs (F) were treated with 40 μM/mL Bay 11- 7082 for 12 h. Cells were then treated with or without 15 mU/mL Hep III for 15/30 min. The levels of ZO-1 were quantified by western blot. (G) A schematic pattern. SDX protects vascular permeability via glycocalyx remodeling against sepsis in vivo and in vitro. Data were expressed as the means ± SEM; *p < 0.05, **p <0 .01.

Figure 5

Inhibition of SDC1 shedding by Sulodexide improved lung injury and survival in septic mice. (A) Pattern diagram of the septic mouse model. Mice were pretreated with sulodexide (40 mg/kg) for 2 h before LPS injection or CLP procedure and those mice were observed for 12 h following LPS and 24 h following CLP before euthanasia. (B, D) ELISA to detect the plasma levels of SDC1 and IL-6 in the LPS (20 mg/kg)-challenged (B) and CLP-induced (D) septic model. (C, E) Survival curves after sulodexide administration to LPS (30 mg/kg)-challenged (C) and CLP-induced (E) mice, n=10/group/experiment. (F) Representative lung tissue sections stained with HE at ×100 (D) in LPS-induced sepsis model. (G) Lung tissue W/D weight ratio in LPS- induced sepsis model. (H) Representative immunofluorescence images of SDC1 in lungs, scale bar = 200 μm. (I) Densitometry of SDC1 immunofluorescence in lungs. Data are expressed as means ± SEM; *p < 0.05.