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X. Wang et al.
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Figure 7. TME5C ameliorates SOS in mice.
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C57BL/6 mice were randomly divided into three groups (n=16 in each group): untreated mice without BMT were defined as the control group, BMT recipient mice treated with vehicle PBS were defined as the BMT group, and BMT recipient mice treated with TME5C were defined as the BMT+TME5C group. (A). Schematic dia- gram of SOS model with the schedule of treatment. (B). Liver macroscopic picture of mice. On day 7 after BMT, some mice were sacrificed, and their livers were pho- tographed. The images show representa- tive livers of mice in each group. (C). Volume of ascites. On day 7 after BMT, ascites was harvested from mice (n=3 per group) and weighed. (D). Plasma levels of liver enzymes. On days 7, 14, and 20 after BMT, blood was collected from mice (n=3 per group), and plasma levels of AST and ALT were measured. (E). Survival of mice. Survival of mice was monitored every day. (F, G, I). H&E, IHC, and Masson staining (H&E and Masson x100, IHC x200). On days 7, 14, and 20 after BMT, some mice were sacrificed. Livers were removed and fixed with formaldehyde solution for H&E staining (blue arrow shows necrosis of hepatocytes and obstruction of liver sinu- soid). Some liver slices were incubated with primary pan-endothelial cell mono- clonal antibody (MECA-32) for IHC stain- ing. Yellow arrows indicate the sinusoidal ECs. Masson staining was also carried out to evaluate the sinusoidal fibrosis of liver. Blue indicates collagen. (H). Quantification of MECA-32 positive-stained sinusoidal ECs in each group (n=3 in each group). (J). SOS score. Scoring of light microscopy his- tological slices stained with H&E or Masson stain were blindly evaluated according to the scoring system. (K). TUNEL stain (x400). Apoptosis of hepato- cytes were assessed using an in situ cell death detection kit. Red arrows indicate apoptotic hepatocytes and sinusoidal ECs. (L). ELISA. Plasma was collected for ELISA to measure the concentrations of TM, FDP, and PAI-1. Results represent the mean ± SD. *P<0.05. BU: busulfan; CY: cyclophos- phamide; BMT: bone marrow transplanta- tion; H&E, hematoxylin-eosin; IHC, immunohistochemistry; TM: thrombomod- ulin; FDP: fibrin degradation product; PAI- 1: plasminogen activator inhibitor-1; PBS: phosphate buffered saline.
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BMT recipients treated with TME5C (Figure 7B-D). Strikingly, 9 out of 16 (56.3%) BMT recipient mice treated with PBS died by day 30 after BMT (Figure 7E), while none of the BMT recipient mice treated with TME5C died during the experimental period (Figure 7E).
Pathological examination of livers removed from BMT mice treated with PBS on day 7 showed severe hemor- rhagic necrosis with partial obstruction of liver sinusoids, infiltration of inflammatory cells, subendothelial hemor- rhage, and loss of liver sinusoidal lining cells (Figure 7F). IHC staining of ECs with MECA-32 indicated a decrease in the number of sinusoid ECs, loss of integrity of sinu- soid walls, and liver sinusoidal EC detachment (Figure 7G,H). These liver abnormalities were partly recovered by the 20th day after BMT in PBS-treated mice. In addi-
tion, Masson staining demonstrated massive sinusoidal fibrosis with collagen deposition in livers removed from BMT recipients treated with PBS (Figure 7I). Importantly, all liver damage and associated findings were less severe in BMT recipients treated with TME5C. Careful patho- logical examination of livers with the SOS scoring system also found that the severity of SOS was significantly less in BMT recipients treated with TME5C than in those treated with PBS throughout the experimental period (Figure 7J). Moreover, TUNEL assays identified fewer apoptotic hepatocytes and sinusoid ECs in livers removed from TME5C-treated BMT mice compared with livers removed from BMT recipients treated with PBS (Figure 7K).
We additionally measured plasma levels of TM, FDP,
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