Liposomal nanocarriers of preassembled glycocalyx expeditiously restore endothelial glycocalyx in endotoxemia

Abstract

The endothelial glycocalyx (EG) is degraded early during sepsis, and currently available treatments are not effective in promptly restoring it. Here, we created liposomal nanocarriers of preassembled glycocalyx (LNPG) by synthesizing glycosylated syndecan-1 and inserting it into the lipid membrane of unilamellar liposomes. We hypothesized that LNPG would fuse with the endothelial cells where EG is degraded and restore EG in sepsis. We induced endotoxemia in C57BL/6J mice using lipopolysaccharides (LPS) and treated them with LNPG, saline, syndecan-1, or liposomes. LNPG significantly prolonged the survival time of LPS-treated mice compared with the other treatments. Immunostaining of en face mesenteric arteries of LPS-treated mice showed that syndecan-1 was fully restored after LNPG administration. In addition, EG height in microvasculature of mouse cremaster muscle was monitored using sidestream dark field imaging. LNPG restored the perfused boundary region (PBR), which is inversely related to EG dimensions, to the control level after LPS administration. Furthermore, flow-induced dilation in isolated mouse mesenteric arterioles was fully recovered after LNPG treatment in LPS-treated mice. In summary, our findings provide evidence of the therapeutic efficacy of LNPG in the LPS-induced mouse model of sepsis, achieved by expeditiously restoring EG through fusion of LNPG with the endothelial plasma membrane and recovery of endothelial function.NEW & NOTEWORTHY Vascular endothelial cells represent the first line of exposure to bacterial endotoxins. Here, we propose a novel therapeutic strategy using liposomes to deliver preassembled glycocalyx to vascular endothelial cell surface and consequently restore endothelial glycocalyx (EG). We tested liposomal nanocarriers of preassembled glycocalyx (LNPG) in vivo and ex vivo to establish for the first time their expeditious therapeutic efficacy in improving survival of lipopolysaccharides (LPS)-treated mice, as achieved by the restoration of EG and recovery of endothelial function.

Keywords: endotoxemia; glycocalyx; liposome; microcirculation; syndecan-1.

Figure 1.

Conceptual schema of the novel therapy using liposomal nanocarriers of preassembled glycocalyx (LNPG). Endothelial glycocalyx covers the luminal surface of vascular endothelial cells. In sepsis, endothelial glycocalyx is degraded. Glycosylated syndecan-1 with histidine tag at its COOH-terminus was inserted into lipid membrane of the unilamellar liposomes to create LNPG. As endothelial glycocalyx is degraded in sepsis, LNPG fused with the plasma membranes of the endothelial cells, resulting in endothelial glycocalyx restoration. CS, chondroitin sulfate; HA, hyaluronic acid; His-tag, 6×-histidine tag; HS, heparan sulfate.

Figure 2.

A: electron microscopy image of unilamellar liposomes. B: mouse full-length syndecan-1 stained with Coomassie blue dye. C: correct incorporation rate of syndecan-1 into the liposomes by means of detergent-mediated reconstitution. D and E: immunostaining of liposomal nanocarriers of preassembled glycocalyx (LNPG) for syndecan-1 (red) and 6×-histidine (green) in permeabilized liposomes. F: merged image of D and E. G and H: immunostaining of LNPG for syndecan-1 (red) and 6×-histidine (green) in nonpermeabilized liposomes. I: merged image of G and H. D–I, insets: magnified images of selected areas. The scale bar equals 20 mm.

Figure 3.

A: Kaplan–Meyer survival curves of male mice in response to lipopolysaccharides (LPS)-induced sepsis with different treatments [liposomal nanocarriers of preassembled glycocalyx (LNPG), syndecan-1, liposomes, or saline]. Treatments were received 1 h after intraperitoneal injection of LPS (15 mg/kg). Numbers of mice are shown in parentheses. B: detailed log-rank tests of A. Mice treated with LNPG survived significantly longer than other groups based on the measurements of median survival time. The surviving rate in LNPG group was 29.4%. There were no significant differences in median survival time and surviving rate among curves of LPS + saline, LPS + syndecan-1, and LPS + liposomes.

Figure 4.

Representative immunostaining of endothelial syndecan-1 (red) and 6×-histidine (green) in en face prepared mesenteric arteries of mice. A: syndecan-1 was observed on the endothelial cells of control mice. B: 6×-histidine tag was not detected in permeabilized samples from the untreated control mice receiving liposomal nanocarriers of preassembled glycocalyx (LNPG). C: expression of syndecan-1 was much decreased in lipopolysaccharides (LPS) + saline group. D and E: syndecan-1 and 6×-histidine staining in LPS + syndecan-1 group with permeabilized samples. F: merged image of D and E. G and H: syndecan-1 and 6×-histidine staining in LPS + LNPG group with permeabilized samples. I: merged image of G and H to show the colocalization of syndecan-1 and 6×-histidine tag. J and K: syndecan-1 and 6×-histidine staining in LPS + LNPG group with nonpermeabilized samples. L: merged image of J and K. M: summarized fluorescence intensity of syndecan-1 in normal vessels (control) and after LPS plus different treatments, indicating that LNPG treatment, not syndecan-1 alone, was capable of fully restoring syndecan-1 expression. N: comparison of 6×-histidine fluorescence intensity of LPS + LNPG group between permeabilized and nonpermeabilized samples. Blue, nuclear staining with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar equals 20 mm. Data are summarized from 18 measurements (6 en face preparations/mouse and 3 mice/group), represented as means ± SE and analyzed by one-way ANOVA. ***P < 0.001.

Figure 5.

A: timeline of GlycoCheck measurements. After inserting jugular vein catheter and preparation for video microscopy of cremaster muscle microcirculation, video images at baseline and after the 1st and 2nd intravenous injections at 30-, 60-, and 90-min time points were recorded. B: representative image of side-stream dark field image of mouse cremaster muscle. C: vessels are automatically recognized and analyzed by the GlycoCheck software. Green lines are valid vascular segments selected for analysis, and yellow lines are invalid segments. D: time-dependent changes in perfused boundary region (PBR). PBR in the vessels with diameters of 4–9 µm were averaged to monitor endothelial glycocalyx (EG) of the capillaries. PBR measured at baseline was used to normalize PBR changes for all data points. PBR increased significantly 30 min after 2nd injection in lipopolysaccharides (LPS) + saline group, which indicated the significant degradation of endothelial glycocalyx. The increase in PBR was significantly higher in LPS + saline and LPS + syndecan-1 groups compared with LPS + liposomal nanocarriers of preassembled glycocalyx (LNPG) group (P < 0.01). PBR changes in LPS + syndecan-1 group were similar to control group. There is no significant difference in PBR between the control (normal mice) and LPS + LNPG groups. E: absolute number of PBR was stable throughout experiment in control group. F–H: time-dependent changes of PBR in LPS + saline, LPG + syndecan-1, and LPS + LNPG groups are shown, respectively. Data in D are represented as means ± SE, and data in E–H are represented as box and whisker plot. Data in D were analyzed by two-way ANOVA; other panels were analyzed by one-way ANOVA. Number of mice was 5e in each group. Every dot represents an averaged PBR from each recording. Each time point consists of 24 recordings/mouse. *P < 0.05, **P < 0.01, and ***P < 0.001 between indicated time points.

Figure 6.

Acetylcholine- (A), NO- (B), and flow-induced dilation (D) in isolated, cannulated, and pressurized mesenteric arteries of mice. Arteries were isolated specifically at the early stage of lipopolysaccharides (LPS)-induced endotoxemia (1 h after LPS administration) to minimize the alteration in cGMP-mediated dilations. The data show that there were no differences in acetylcholine- and NO-mediated dilations among groups. In study of flow-induced dilation, an initial shear stress of 10 or 20 dyn/cm² was applied to each vessel by adjusting the intraluminal flow calculated according to the basal diameter obtained before the onset of the intraluminal flow. In LPS + saline group, flow-induced dilation was reduced by 38.6 and 26.8% in response to 10 and 20 dyn/cm², respectively. Liposomal nanocarriers of preassembled glycocalyx (LNPG) treatment, but not syndecan-1 or liposomes treatment, fully recovered flow-induced dilation. C: the representative traces of vessel diameter in flow-induced dilation for control, LPS + saline, and LPS + LNPG group. Values are means ± SE (n = 4 mice/group). Data were analyzed by one-way ANOVA. *P < 0.05, **P < 0.01. ACh, acetylcholine; lipo, liposomes; NO, nitric oxide; PD, passive diameter; sdc1, syndecan-1.