Hiroyuki Yuasa

Bacterial lipopolysaccharide decreases thrombomodulin expression in the sinusoidal endothelial cells of rats – a possible mechanism of intrasinusoidal microthrombus formation and liver dysfunction

BACKGROUND/AIMS To elucidate the mechanism of liver dysfunction occurring in patients with sepsis, we evaluated the effect of bacterial lipopolysaccharide (LPS) on the expression of thrombomodulin (TM) in rat sinusoidal endothelial cells (SECs) and the therapeutic efficacy of exogenous recombinant TM. METHODS We induced endotoxemia in rats by bolus intraperitoneal injection of LPS. TM antigen levels within tissues were assessed by immunohistochemistry. We measured TM in cultured SECs by enzyme immunoassay, functional analysis and real-time polymerase chain reaction (PCR). RESULTS TM antigen and activity levels were significantly decreased in SECs isolated from LPS-treated rats after 3 and 6 h treatment, and recovered after 12 h treatment, correlating with immunohistochemical observations. In contrast, TM messenger RNA was decreased after 6 and 12 h treatment, and slightly recovered after 24 h treatment. TM expression in cultured SECs isolated from normal rats was also reduced after treatment with LPS and tumor necrosis factor (TNF)-alpha in vitro. The increased levels of serum fibrin degradation products (FDP), fibrin deposition within liver sinusoids, injury of SECs and liver dysfunction induced by LPS in our rat model was improved by recombinant TM treatment. CONCLUSIONS Decreased TM expression in SECs of LPS-treated rats may result in intrasinusoidal microthrombus formation and subsequent liver dysfunction during sepsis.

Molecular cloning, tissue distribution and androgen regulation of rat protein C inhibitor

Protein C inhibitor (PCI) is the plasma serine protease inhibitor of activated protein C, the active enzyme of the anticoagulant protein C pathway. Recently, PCI was also detected in human seminal plasma and reproductive organs (testis, seminal vesicle and prostate) suggesting that PCI may also play an important role in the reproductive system. In this study, we cloned the full length of rat PCI cDNA, and determined its amino acid sequence and tissue distribution. We also evaluated the effect of androgen on PCI mRNA expression in seminal vesicles and testes. The isolated 2074‐bp rat PCI cDNA was composed of a 47‐bp 5′‐non‐coding region, a 1218‐bp coding region of a 406‐amino acid precursor protein, a stop codon and a 806‐bp 3′‐non‐coding region. The deduced amino acid sequence of rat PCI showed 85.7%, 64.1% and 62.2% homology with that of mouse, rhesus monkey and human PCIs, respectively. Northern blot analysis showed that the rat PCI mRNA is expressed strongly in the seminal vesicle, moderately in the testis, but not in the liver. PCI mRNA expression in seminal vesicles and testes was found to increase during the process of development, suggesting that it is under androgen control. Subsequently, we examined the effect of castration and/or treatment with 17β‐estradiol or testosterone on PCI mRNA expression in the mature rat seminal vesicles. The PCI mRNA expression in seminal vesicles was significantly decreased after castration or 17β‐estradiol treatment. Testosterone itself did not affect PCI mRNA expression, but treatment in castrated rats significantly enhanced its mRNA expression. These findings suggest that the PCI gene expression in rat seminal vesicles is regulated by androgen.

Lipopolysaccharide‐induced decreased protein S expression in liver cells is mediated by MEK/ERK signaling and NFκB activation: involvement of membrane‐bound CD14 and toll‐like receptor‐4

Summary.  Background: The vitamin K‐dependent protein S (PS), mainly synthesized in hepatocytes and endothelial cells, plays a critical role in the anticoagulant activity of plasma. The decreased plasma level of PS in sepsis is associated with thrombotic tendency, but the mechanism is unclear. Objectives: In the present study, we examined the effect of lipopolysaccharide (LPS) on PS expression in vivo in rat liver, and in vitro in isolated hepatocytes and sinusoidal endothelial cells (SECs) from normal rats. Results: LPS induced a progressive decrease of plasma PS antigen level up to 12 h with a slight recovery at 24 h, and a transient decrease of liver PS mRNA level at 4–8 h with a complete recovery at 24 h. In the in vitro studies, LPS decreased PS antigen and mRNA levels in both hepatocytes and SECs. After LPS treatment, tumor necrosis factor‐α (TNF‐α), interleukin‐6 (IL‐6) and interferon‐γ (IFN‐γ) transiently increased in plasma. IL‐6 increased the protein expression of PS from hepatocytes, while TNF‐α decreased it from SECs. LPS increased CD14 in hepatocytes and decreased it in SECs, but did not affect toll‐like receptor‐4 (TLR‐4) expression in both cells. Antirat CD14 and antirat TLR‐4 antibodies inhibited LPS‐induced NFκB activation, and a NFκB inhibitor suppressed LPS‐induced decreased PS expression in both cells. Furthermore, MEK inhibitor blocked LPS‐induced decreased PS expression in both cells. Conclusions: These findings suggest that LPS‐induced decreased PS expression in hepatocytes and SECs is mediated by MEK/ERK signaling and NFκB activation and that membrane‐bound CD14 and TLR‐4 are involved in this mechanism. These findings may explain in part the decreased level of plasma PS and thrombotic tendency in sepsis.

Regulatory mechanisms of C4b-binding protein (C4BP)α and β expression in rat hepatocytes by lipopolysaccharide and interleukin-6

Summary.  Background: C4b‐binding protein (C4BP), a multimeric protein structurally composed of α chains (C4BPα) and a β chain (C4BPβ), regulates the anticoagulant activity of protein S (PS). Patients with sepsis have increased levels of plasma C4BP, which appears to be induced by interleukin (IL)‐6. However, it is not fully understood how lipopolysaccharide (LPS) and IL‐6 affect the plasma C4BP antigen level and C4BPα and C4BPβ expression in hepatocytes. Objectives: To assess the effect of LPS and IL‐6 on plasma C4BP, PS–C4BP complex levels, PS activity, and C4BP expression by rat liver in vivo and on C4BP expression by isolated rat hepatocytes in vitro. Results: Plasma C4BP antigen level transiently decreased from 2 to 12 h after LPS (2 mg kg−1) injection, and then it abruptly increased up to 24 h after LPS injection. Plasma C4BP antigen level increased until 8 h after IL‐6 (10 μg kg−1) injection, and then gradually decreased up to 24 h after IL‐6 injection. LPS significantly decreased the protein and mRNA expression of both C4BPα and C4BPβ in rat hepatocytes, and this effect was inhibited by NFκB and MEK/ERK inhibitors. IL‐6 mediated increase in C4BPβ expression in rat hepatocytes, which leads to increased plasma PS–C4BP complex level and to decreased plasma PS activity, was inhibited by inhibition of STAT‐3. Conclusion: LPS decreases both C4BPα and C4BPβ expression via the NFκB and MEK/ERK pathways, whereas IL‐6 specifically increases C4BPβ expression via the STAT‐3 pathway, causing an increase in plasma PS–C4BP complex, and thus decreasing the anticoagulant activity of PS.