Sphingosine 1-phosphate enhances portal pressure in isolated perfused liver via S1P2 with Rho activation

doi:10.1016/j.bbrc.2004.04.207

Biochemical and Biophysical Research Communications

Volume 320, Issue 3, 30 July 2004, Pages 754-759

https://doi.org/10.1016/j.bbrc.2004.04.207 Get rights and content

Abstract

Although structural changes are most important to determine vascular resistance in portal hypertension, vasoactive mediators also contribute to its regulation. Hepatic stellate cells (HSCs) are assumed to play a role in modulating intrahepatic vascular resistance based on their residence in the space of Disse and capacity to contract. Because sphingosine 1-phosphate (S1P) has been shown to stimulate HSC contractility, we wondered if S1P could regulate portal pressure. S1P at 0.5–5 μM increased portal pressure in isolated rat perfused liver. This effect was abrogated in the presence of a binding antagonist for S1P₂, JTE-013. Perfusion of isolated rat liver with 5 μM S1P increased Rho activity in the liver, and co-perfusion with JTE-013 cancelled S1P-induced Rho activation. Because S1P is present in human plasma at approximately 0.2 μM, S1P might readily regulate portal vascular tone in physiological and pathological status. The antagonist for S1P₂ merits consideration for treatment of portal hypertension.

Section snippets

Materials and methods

Materials. S1P was obtained from BIOMOL Research Laboratories, anti-RhoA (26C4) was from Santa Cruz Biotechnology, and endothelin-1 was from Calbiochem. JTE-013 was kindly provided by Central Pharmaceutical Research Institute, Japan Tobacco (Osaka, Japan).

Animals. Male Sprague–Dawley rats (Shizuoka Laboratory Animal Center, Shizuoka, Japan) were fed a standard pelleted diet and water ad libitum, and used in all the experiments. All animals received human care in compliance with the

Effect of S1P on portal pressure

Before examining the effects of test materials on portal pressure in isolated rat liver, the perfusion with KHB alone was continued for approximately 10–15 min to obtain the stable portal pressure; the range of the stable, essentially unaltered pressure was between 27 and 34 cm H₂O in our experiment. Fig. 1A demonstrates the representative result of perfusion experiment of isolated rat liver with S1P, the concentration of which was serially increased, suggesting that S1P may enhance portal

Discussion

Isolated perfused liver in rats has long been used to examine regulators of portal pressure [31], though it has the limitation of non-physiological status. For example, the hepatic artery is ligated when starting perfusion [31] and subsequently rats are sacrificed, indicating that the contribution of arterial pressure to portal one is not under consideration. Thus, the rationale of using this model in clarifying regulators of portal pressure is based on the fact that resistance of portal blood

Acknowledgements

The authors thank Central Pharmaceutical Research Institute, Japan Tobacco Inc., for valuable materials.

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Knowing the pathophysiology of portal hypertension can help to develop effective therapeutic strategies. One study elucidated that S1P increases portal pressure, and pretreatment with S1PR2 antagonists abolishes S1P-induced enhancement in portal pressure in isolated perfused rat liver, indicating that S1PR2 antagonists may be considered a potential strategy for portal pressure treatment [81]. A previous study verified that inhibiting Sphks and S1PL reduced proliferation, invasion and migration, while upregulating Sphks and S1PL enhanced proliferation in HCC cell lines.
Liver dysfunction is always accompanied by lipid metabolism dysfunction. Apolipoprotein M (apoM), a member of the apolipoprotein family, is primarily expressed and secreted from the liver. apoM is the main chaperone of sphingosine-1-phosphate (S1P), a small signalling molecule associated with numerous physiologic and pathophysiologic processes. In addition to transport, apoM also influences the biologic effects of S1P. Most recently, numerous studies have investigated the potential role of the apoM-S1P axis in a variety of hepatic diseases. These include liver fibrosis, viral hepatitis B and C infection, hepatobiliary disease, non-alcoholic and alcoholic steatohepatitis, acute liver injury and hepatocellular carcinoma. In this review, the roles of apoM and S1P in the development of hepatic diseases are summarized, and novel insights into the diagnosis and treatment of hepatic diseases are discussed.
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Thus, it has been speculated that hepatic stellate cells may play a direct role in the pathogenesis of portal hypertension, one of major complications of liver fibrosis. S1P was shown to enhance contractility of hepatic stellate cells in vitro [80], and further to increase portal vein pressure ex vivo in rats with Rho and Rho kinase activation via S1P2 [92]. Furthermore, it has been recently shown that the intravenous administration of S1P2 antagonist, JTE-013, promptly reduces portal vein pressure with Rho kinase inactivation in rats with portal hypertension caused by bile duct ligation [86].
Fibrosis is a pathological process characterized by massive deposition of extracellular matrix (ECM) such as type I/III collagens and fibronectin that are secreted by an expanded pool of myofibroblasts, which are phenotypically altered fibroblasts with more contractile, proliferative, migratory and secretory activities. Fibrosis occurs in various organs including the lung, heart, liver and kidney, resulting in loss of normal tissue architecture and functions. Myofibroblasts could originate from multiple sources including tissue-resident fibroblasts, epithelial and endothelial cells through mechanisms of epithelial/endothelial-mesenchymal transition (EMT/EndMT), and bone marrow-derived circulating progenitors called fibrocytes. Emerging evidence in recent years shows that sphingosine-1-phosphate (S1P) acts on several types of target cells and is engaged in pro-fibrotic inflammatory process and fibrogenic process through multiple mechanisms, which include vascular permeability change, leukocyte infiltration, and migration, proliferation and myofibroblast differentiation of fibroblasts. Many of these S1P actions are receptor subtype-specific. In these actions, S1P has multiple cross-talks with other cytokines, particularly transforming growth factor-β (TGFβ), which plays a major role in fibrosis. The cross-talks include the regulation of S1P production through altered expression and activity of sphingosine kinases in fibrotic lesions, altered expression of S1P receptors, and S1P receptor-mediated transactivation of TGFβ signaling pathway. These cross-talks may give rise to a feed-forward, amplifying loop between S1P and TGFβ, and possibly with other cytokines in stimulating fibrogenesis. Another lysophospholipid mediator lysophosphatidic acid has also been recently implicated in fibrosis. The lysophospholipid signaling pathways represent novel, promising therapeutic targets for treating refractory fibrotic diseases. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.
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Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were assayed by standard enzymatic methods. Portal pressure in isolated perfused rat livers was measured according to previously described procedures, with some modifications [21]. The perfusion with Krebs–Henseleit buffer (Sigma–Aldrich Inc.) was continued until the monitored inlet pressure value became stable at a constant flow rate of 0.3 ml min−1 liver volume−1 (ml).
Hepatic stellate cells are thought to play a role in modulating intrahepatic vascular resistance based on their capacity to contract via Rho signaling. We investigated the effect of a Rho-kinase inhibitor on ischemia–reperfusion injury in the steatotic liver.
Steatotic livers, induced by a choline-deficient diet in rats, were subjected to ischemia–reperfusion injury. Hepatic stellate cells isolated from steatotic livers were analyzed for contractility and Rho signaling activity. The portal pressure of the perfused rat liver and the survival rate after ischemia–reperfusion were also investigated.
Hepatic stellate cells from steatotic livers showed increased contractility and upregulation of Rho-kinase 2 compared with those from normal livers. Furthermore, endothelin-1 significantly enhanced the contractility and phosphorylation level of myosin light chain and cofilin in hepatic stellate cells isolated from steatotic livers. A specific Rho-kinase inhibitor, fasudil, significantly suppressed the contractility and decreased the phosphorylation levels of myosin light chain and cofilin. Serum levels of endothelin-1 were markedly increased after IR in rats with steatotic livers, whereas fasudil significantly decreased endothelin-1 serum levels. Rats with steatotic livers showed a significant increase in portal perfusion pressure after ischemia–reperfusion and a significant decrease in survival rate; fasudil treatment significantly reduced these effects.
Activation of Rho/Rho-kinase signaling in hepatic stellate cells isolated from steatotic livers is associated with an increased susceptibility to ischemia–reperfusion injury. A Rho-kinase inhibitor attenuated the activation of hepatic stellate cells isolated from steatotic livers and improved ischemia–reperfusion injury in steatotic rats.
Plasma concentration of bioactive lipid mediator sphingosine 1-phosphate is reduced in patients with chronic hepatitis C
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We have been focusing our attention on the potential role of S1P in liver pathophysiology. We first demonstrated that S1P stimulates proliferation and contractility via S1P2 in rat hepatic stellate cells [27,28]. We also revealed that S1P inhibits proliferation in rat hepatocytes with Rho activation via S1P2 and that the administration of S1P in 70% hepatectomized rats reduces the synthesis of DNA in hepatocytes [29].
Bioactive lipid mediator S1P has been suggested to play pathophysiological roles in various fields of clinical science as a circulating paracrine mediator. We previously established a reliable method of measuring plasma S1P concentration, and reported that the one in healthy subjects has a gender difference and a correlation with red blood cell (RBC)-parameters, however, the reports of S1P measurements in the blood in patients with a specific disease have been scarce. Because our previous evidence suggests that S1P is involved in liver pathophysiology, we examined plasma S1P concentration in chronic hepatitis C patients.
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Sphingosine 1-phosphate regulates regeneration and fibrosis after liver injury via sphingosine 1-phosphate receptor 2
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Sphingosine 1-phosphate (S1P), a bioactive lipid mediator, stimulates proliferation and contractility in hepatic stellate cells, the principal matrix-producing cells in the liver, and inhibits proliferation via S1P receptor 2 (S1P₂) in hepatocytes in rats in vitro. A potential role of S1P and S1P₂ in liver regeneration and fibrosis was examined in S1P₂-deficient mice. Nuclear 5-bromo-2′-deoxy-uridine labeling, proliferating cell nuclear antigen (PCNA) staining in hepatocytes, and the ratio of liver weight to body weight were enhanced at 48 h in S1P₂-deficient mice after a single carbon tetrachloride (CCl₄) injection. After dimethylnitrosamine (DMN) administration with a lethal dose, PCNA staining in hepatocytes was enhanced at 48 h and survival rate was higher in S1P₂-deficient mice. Serum aminotransferase level was unaltered in those mice compared with wild-type mice in both CCl₄- and DMN-induced liver injury, suggesting that S1P₂ inactivation accelerated regeneration not as a response to enhanced liver damage. After chronic CCl₄ administration, fibrosis was less apparent, with reduced expression of smooth-muscle α-actin-positive cells in the livers of S1P₂-deficient mice, suggesting that S1P₂ inactivation ameliorated CCl₄-induced fibrosis due to the decreased accumulation of hepatic stellate cells. Thus, S1P plays a significant role in regeneration and fibrosis after liver injury via S1P₂.
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Sphingosine 1-phosphate enhances portal pressure in isolated perfused liver via S1P2 with Rho activation

Abstract

Section snippets

Materials and methods

Effect of S1P on portal pressure

Discussion

Acknowledgements

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Hepatology

Gene

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FEBS Lett.

Eur. J. Pharmacol.

J. Biol. Chem.

Eur. J. Pharmacol.

J. Hepatol.

Gastroenterology

Verapamil favorably influences hepatic microvascular exchange and function in rats with cirrhosis of the liver

J. Clin. Invest.

Sphingolipid metabolism and cell growth regulation

FASEB J.

Sphingosine 1-phosphate enhances portal pressure in isolated perfused liver via S1P₂ with Rho activation