Volume 38, Issue 1, Pages 9-17 (January 2003)

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Volume 38, Issue 1, Pages 9-17 (January 2003) Bacterial lipopolysaccharide decreases thrombomodulin expression in the sinusoidal endothelial cells of rats – a possible mechanism of intrasinusoidal microthrombus formation and liver dysfunction Masane Kume, Tatsuya Hayashi, Hiroyuki Yuasa, Hitoshi Tanaka, Junji Nishioka, Masaru Ido, Esteban C. Gabazza, Yoshifumi Kawarada, Koji Suzuki Journal of Hepatology Volume 38, Issue 1, Pages 9-17 (January 2003) DOI: 10.1016/S0168-8278(02)00324-0

Fig. 1 Immunohistochemical analysis of TM expression in the rat liver before and after LPS treatment. Tissue-Tek-embedded 6-μm liver sections prepared from normal rats (A); or rats 6 h after LPS treatment (B) were stained by ENVISION kit/HRP (AEC) using anti-rat TM rabbit IgG. Adjacent sections were stained with hematoxylin. (Original magnification: ×400). Journal of Hepatology 2003 38, 9-17DOI: (10.1016/S0168-8278(02)00324-0)

Fig. 2 TM activity and TM mRNA levels in SECs isolated from LPS-treated rats. SECs isolated from rats treated with LPS for variable periods were cultured for 12 h in the presence of fetal calf serum. TM activity (A) was determined from the protein C-activating cofactor activity. TM mRNA levels (B) were determined by real-time PCR. Representative data are shown. *P<0.05 versus time 0. Journal of Hepatology 2003 38, 9-17DOI: (10.1016/S0168-8278(02)00324-0)

Fig. 3 The effect of LPS on TM expression in SECs isolated from normal rats. SECs isolated from normal rats were treated for 6 h with the indicated concentrations of LPS in the presence of fetal calf serum. TM activity (A) was determined from the protein C-activating cofactor activity. TM mRNA level (B) in the cells was determined by real-time PCR. The relative TM mRNA levels in LPS-treated SECs were obtained by comparison with TM mRNA levels in untreated cells. *P<0.05 versus LPS (0 μg/ml). Journal of Hepatology 2003 38, 9-17DOI: (10.1016/S0168-8278(02)00324-0)

Fig. 4 Changes of plasma cytokine levels in LPS-treated rats. Plasma levels of TNF-α (closed circle), IL-6 (closed triangle) and IFN-γ (open circle) in LPS-treated rats were determined by specific enzyme immunoassay. Data are expressed as the mean±SD (n=4). *P<0.05 versus time 0. Journal of Hepatology 2003 38, 9-17DOI: (10.1016/S0168-8278(02)00324-0)

Fig. 5 The effect of TNF-α, IL-6 and IFN-γ on TM expression in SECs isolated from normal rats. SECs isolated from normal rats were treated for 12 h with the indicated concentrations of TNF-α (A); IL-6 (B); and IFN-γ (C) in the absence of fetal calf serum. TM activity was determined from the protein C-activating cofactor activity. *P<0.05 versus TNF-α (0 IU/ml). Journal of Hepatology 2003 38, 9-17DOI: (10.1016/S0168-8278(02)00324-0)

Fig. 6 Effect of recombinant TM on serum FDP levels and thrombus formation in sinusoids of LPS-treated rats. (A) Serum FDP levels were determined at each time point by enzyme immunoassay for rat Fbg for LPS-treated rats (open bar) and LPS-treated rats pre-treated with recombinant TM (closed bar). Data are expressed as the mean±SD (n=3). *P<0.05 versus without recombinant TM. (B) Intrasinusoidal fibrin within liver tissue sections prepared from LPS-treated rats and LPS-treated rats pre-treated with recombinant TM were detected by ENVISION kit/HRP (AEC) using anti-rat Fbg rabbit IgG. (Magnification: ×400). Journal of Hepatology 2003 38, 9-17DOI: (10.1016/S0168-8278(02)00324-0)

Fig. 7 Effect of recombinant TM on injury of SECs and liver dysfunction in LPS-treated rats. Plasma HA antigen levels (A); and ALT activity (B) were determined at each time point by enzyme immunoassay and Kodak Ektachem 250 automated analyzer, respectively, for citrated plasma samples obtained from LPS-treated rats (open bar) and LPS-treated rats pre-treated with recombinant TM (closed bar). Data are expressed as the mean±SD (n=3). *P<0.05 versus without recombinant TM. Journal of Hepatology 2003 38, 9-17DOI: (10.1016/S0168-8278(02)00324-0)

Fig. 8 Effect of recombinant TM on plasma cytokine levels in LPS-treated rats. Plasma TNF-α levels (A); and IL-6 levels (B) were determined at each time point by specific enzyme immunoassay in citrated plasma samples obtained from LPS-treated rats (open bar) and LPS-treated rats pre-treated with recombinant TM (closed bar). Data are expressed as the mean±SD (n=3). *P<0.05 versus without recombinant TM. Journal of Hepatology 2003 38, 9-17DOI: (10.1016/S0168-8278(02)00324-0)