Fig 2: Effect of Nef on endothelial glycocalyx degradation in LPS-induced ARDS. Mice were treated with LPS (20 mg/kg) for 6 h, pretreated with Nef (20 mg/kg/day) for 7 days followed by LPS (20 mg/kg) for 6 h, or pretreated with Nef (20 mg/kg/day) alone for 7 days (n = 6 mice/group). The content of HS (red) (A) and SDC-1 (red) (B) in mice lungs were determined by detected by immunofluorescence. Thrombomodulin / BDCA-3 was used as an endothelial cell marker. (magnification 200×, scale bar: 50 µm). (C,E) Fluorescence intensity analyses of (A,B), respectively. The serum levels of HS (D) and SDC-1 (F) in mice were detected by ELISA kits. HUVECs were treated with LPS (1 µg/mL) for 6 h, Nef (10 µM) for 1 h followed by LPS (1 µg/mL) for 6 h, or Nef (10 µM) alone for 7 h. The content of HS (green, magnification 100×, scale bar: 100 µm) (G) and SDC-1 (red, magnification 200×, scale bar: 50 µm) (H) in HUVECs detected by immunofluorescence. (I,K) Fluorescence intensity analyses of (G,H), respectively. The levels of shed HS (J) and SDC-1 (L) in HUVECs culture medium were determined by ELISA kits. Data were presented as mean ± SD for three independent experiments. #p < 0.05 compared with the control group; *p < 0.05 compared with the LPS group.

Fig 3: Effect of heparin on endothelial glycocalyx degradation products in an LPS-induced sepsis rat model. (A) HS and (C) SDC-1 in plasma was determined using an ELISA at 0.5, 2 and 6 h after LPS injection. Effect of UFH or NAH on (B) HS and (D) SDC-1 in plasma at 2 and 6 h after LPS injection. The distribution of (E) HS (red) and (F) SDC-1 (red) in rat lungs was assessed using staining with specific antibodies. Endothelial cell marker of pulmonary vascular was assessed using BDCA-3 (green) staining (magnification, ×200; scale bar, 50 µm). (G) Fluorescence intensity analyses of the images presented in (E). (H) Fluorescence intensity analyses of the images presented in (F). Data are presented as mean ± standard deviation for 3 independent experiments. *P<0.05 vs. the control group; #P<0.05 vs. the LPS group. The HS and SDC-1 data were determined using a one-way analysis of variance followed by a Student-Newman-Keuls test for multiple group comparisons. LPS, lipopolysaccharide; HS, heparan sulphate; SDC-1, syndecan-1; UFH, unfractionated heparin; NAH, N-acetylheparin; BDCA-3, thrombomodulin.