Sphingosine 1-phosphate protects rat liver sinusoidal endothelial cells from ethanol-induced apoptosis: Role of intracellular calcium and nitric oxide

Authors


  • Potential conflict of interest: Nothing to report.

Abstract

In alcoholic liver disease, ethanol-induced damage to sinusoidal endothelial cells (SECs) appears to be important in the progression of liver damage. However, little is known about the mechanisms responsible for protection of SECs against ethanol-induced injury. To elucidate the role of sphingosine 1-phosphate (S1P), which is stored in platelets and may be released from them on their activation, we investigated the effect of S1P on rat liver SECs in primary culture. Pretreatment of cells with 1 μmol/L S1P attenuated ethanol-induced apoptosis. Electron microscopy confirmed this protective effect of S1P on damaged SECs in liver tissues after perfusion of ethanol. In the absence of ethanol, S1P increased DNA synthesis as determined via incorporation of bromodeoxyuridine. S1P also ameliorated the decreased DNA synthesis of cells induced by ethanol. Addition of S1P to cells induced an increase in intracellular calcium concentrations and NO production in cells. Western blotting revealed that S1P significantly induced the activation of endothelial NO synthase (eNOS), but not Akt, and that S1P-induced activation of eNOS was blocked by trifluoperazine, a calmodulin inhibitor. Furthermore, NG-nitro-L-arginine methyl ester, a NO synthase inhibitor, cancelled the effect of S1P on DNA synthesis, apoptosis, and NO production in vitro as well as the protective effect of S1P on cell damage in situ. In conclusion, the biological effect of S1P is at least partially mediated by Ca2+-sensitive eNOS activation and subsequent NO formation; extracellular S1P could contribute to sinusoidal protection and remodeling in alcoholic liver injury. (HEPATOLOGY 2006;44:1278–1287.)

In alcoholic liver injury, ethanol-induced perturbation of microcirculation has been extensively studied in association with endotoxins, Kupffer cell activation, endothelium-derived factors, and leukocyte adhesion to sinusoidal endothelial cells (SECs).1–5 The structural and functional alterations of sinusoidal endothelial fenestrae also have been reported to involve impaired microcirculatory exchange of fluids and solutes between the sinusoidal lumen and the space of Disse.6, 7 In experimental and human studies, plasma hyaluronic acid levels are elevated in alcoholic liver injury, which may reflect a diminished hepatic clearance in SECs.8, 9

These findings suggest that SECs play a key role in alcoholic liver disease, in that they are directly exposed to high concentrations of ethanol in sinusoidal blood flow. The importance of SECs in alcoholic liver injury has been further supported by experimental data showing that SECs are chronologically the first hepatic cells to undergo pathological changes,10 although Kupffer cells may be the first to respond to ethanol or lipopolysaccharide (LPS) resulting from alcohol consumption. However, the mechanism underlying the biological regulation of liver endothelium is poorly understood in ethanol-induced liver injury.

The maintenance of the endothelium as a semipermeable barrier has been shown to require circulating blood platelets; in vitro and in vivo models have indicated profound defects in endothelial cell barrier function after perfusion with platelet-poor plasma or after depletion of platelets with antiplatelet antibodies.11 Because these events are completely reversed by the infusion of platelet-rich plasma,12 platelets are suggested to play a crucial role in normal vascular function.

Recently, sphingosine 1-phosphate (S1P), a serum-borne bioactive lipid released from activated platelets,13, 14 has been revealed to regulate diverse biological processes such as cell proliferation,15 migration,16 wound healing,17 and inhibition of apoptosis.18, 19 S1P was originally considered to be formed merely as an intermediate in the detoxification of sphingosine by its phosphorylation and subsequent degradation, but the findings of these biological responses highlight the importance of S1P as a signaling molecule. It is well known that S1P belongs to a class of lipid mediators that function not only inside cells but also as natural ligands for cell-surface receptors, which are members of the G-protein–coupled family of receptors. These receptors were initially called endothelial differentiation gene receptors and have been renamed as S1P receptors. To date, five specific S1P receptors (S1P1 [EDG-1], S1P2 [EDG-5], S1P3 [EDG-3], S1P4 [EDG-6], and S1P5 [EDG-8]) have been identified, and these are coupled to different intracellular second messenger systems, including adenylate cyclase, phospholipase C, phosphatidylinositol 3-kinase (PI-3K)/Akt/endothelial NO synthase (eNOS), mitogen-activated protein kinases, as well as Rho- and Ras-dependent pathways.20

These findings provide strong support for the idea that extracellular S1P released from activated platelets could contribute to protection of SECs in ethanol-induced liver injury, because platelets have been implicated to accumulate within the liver early after administration of LPS, which is responsible for the pathophysiology of alcoholic liver disease.14, 21

In this study, we explore the effect of extracellular S1P on rat liver SECs in primary culture and isolated perfused rat liver tissues and provide evidence that S1P contributes to sinusoidal protection against ethanol-induced injury by Ca2+-sensitive eNOS activation and subsequent NO formation.

Abbreviations

SEC, sinusoidal endothelial cell; S1P, sphingosine 1-phosphate; eNOS, endothelial NO synthase; LPS, lipopolysaccharide; PI-3K, phosphatidylinositol 3-kinase; VEGF, vascular endothelial growth factor; GBSS, Gey's balanced salt solution; BrdU, bromodeoxyuridine; TUNEL, terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling; L-NAME, NG-nitro-L-arginine methyl ester; CaM, calmodulin.

Materials and Methods

Materials.

S1P and vascular endothelial growth factor (VEGF) were obtained from Sigma-Aldrich Co. (St. Louis, MO) and Wako Pure Chemical Industries, Ltd. (Osaka, Japan), respectively. Fura-2 acetomethoxy ester (fura-2/AM) and pluronic F127 were purchased from Molecular Probes (Junction City, OR). All other chemicals were of analytical grade.

Preparation of SECs in Primary Culture.

SECs were isolated based on a modification of the method of Braet and Wisse et al.22 Briefly, the livers of female Wistar rats weighing 200-250 g (Charles River Japan Inc., Saitama, Japan) were perfused in situ through the portal vein with Gey's balanced salt solution (GBSS) without calcium at 37°C for 20 minutes at a flow rate of 10 mL/min. The livers were then perfused with GBSS with calcium containing 0.05% collagenase A (Roche Diagnostics Co., Mannheim, Germany) at 37°C for 20 minutes at a flow rate of 5 mL/min. Subsequently, livers were excised and digested by shaking in 100 mL fresh GBSS with calcium containing 0.05% collagenase A, 0.001% DNase (Roche Diagnostics Co.), and 5.3 mL of heat-inactivated fetal bovine serum for 10 minutes at 37°C. The cell suspension was centrifuged at 100g for 5 minutes. The supernatant enriched in SECs was centrifuged for 10 minutes at 350g twice, and the cells were purified by isopycnic sedimentation in a two-step Percoll gradient (25% and 50%) and centrifuged at 900g for 20 minutes. The fraction of SECs was collected and washed in phosphate-buffered saline. SECs were cultured onto collagen-coated dishes in EBM-2 medium containing 20 ng/mL VEGF as previously described.23 The study protocol was approved by the Animal Care Committee of Juntendo University, which conforms to the National Institutes of Health guidelines.

Morphological Characterization of SECs by Scanning Electron Microscopy.

SECs at day 2 in culture were rinsed twice with phosphate-buffered saline and fixed with 2% glutaraldehyde in buffer (0.1 mol/L Na-cacodylate and 0.1 mol/L sucrose; pH 7.4) for 12 hours. Fixed culture samples were subsequently treated with filtered 1% taninic acid in 0.15 mol/L Na-cacodylate (pH 7.4) for 1 hour and postfixed with 1% osmiumtetroxide in 0.1 mol/L Na-cacodylate (pH 7.4) for 1 hour. They were dehydrated in a graded ethanol series and dried with hexamethyldisilazane. The dried specimens were mounted on an aluminum holder and coated with osmium sputter. The samples were examined with a scanning electron microscope (S-800; Hitachi, Tokyo, Japan) at an accelerating voltage of 5 KV.

Incorporation of Bromodeoxyuridine in SECs.

At day 2 in culture, cells were incubated with 10 μmol/mL bromodeoxyuridine (BrdU) (Sigma-Aldrich Co., St. Louis, MO) at 37°C for 1 hour, and fixed with 4% paraformaldehyde overnight at 4°C. BrdU retrieval was performed by DNA denaturation in 0.05% protease (Sigma-Aldrich Co., St. Louis, MO) and 1 N HCl at 37°C for 15 minutes. Subsequently, BrdU was detected using an anti-BrdU antibody (Vector Laboratories, Inc., Burlingame, CA).

Detection of Apoptosis by Terminal Deoxynucleotidyl Transferase–Mediated dUTP Nick End Labeling.

At day 2 in culture, apoptoic cells were determined by a terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) method with a fluorescent apoptosis detection system kit (Promega, Madison, WI). The stained cells were recorded on a fluorescence microscope (Axiophto ZVS 3C75DE; Carl Zeiss, Goleta, CA) using Ektachrome Dyna 400 film (Eastman Kodak, Rochester, NY).

Detection of Apoptosis by Transmission Electron Microscopy.

After incubation at day 2 in culture with 150 mmol/L ethanol for 6 hours, SECs were rinsed twice with phosphate-buffered saline and fixed with 2.5% glutaraldehyde in 0.1 mol/L phosphate buffer for 2 hours. After fixation, samples were rinsed with phosphate-buffered saline and postfixed with 1% osmium tetroxide for 2 hours. Fixed cultures were dehydrated in ethanol and then embedded on a Reichert ULTRACUT (Leica Microsystems, Tokyo, Japan). The samples were stained with uranyl acetate and examined by a transmission electron microscope (H-7100; Hitachi).

Detection of Genomic DNA Fragmentation.

Following incubation of SECs at day 2 in culture with 150 mmol/L ethanol for 6 hours, they were collected and centrifuged at 5,000 rpm for 3 minutes. DNA was extracted from the cells by using an Apoptotic DNA Ladder Kit (Roche Diagnostics Co.). Fragmentation of DNA was confirmed on 1% agarose gels after electrophoresis and staining with ethidium bromide for visualization under ultraviolet transillumination.

Serial Quantitative Analysis of [Ca2+]i in SECs Using a Fluorescence Microscope Imaging System.

Intracellular calcium concentrations ([Ca2+]i) of cells were measured as previously described.24–26 Fura-2 acetomethoxy ester with pluronic F127 was dissolved in EBM-2, in which final concentrations of fura-2 acetomethoxy ester and pluronic F127 were 4 μmol/L and 0.1%, respectively. After loading with fura-2 acetomethoxy ester solution at 37°C for 30 minutes, cells were placed in a fluorescence microscope (Diaphot, Nikon, Tokyo, Japan) with a 100-w xenon arc lamp as a light source.

Detection of NO Formation in Image Analysis System.

Diaminofluorescein-2 diacetate (Daiichi Pure Chemicals Co., Ltd, Japan), a fluorescent NO indicator,27 was dissolved in fetal bovine serum–free EBM-2 medium to a concentration of 10 μmol/L before experiment. SECs were cultured onto a 35-mm dish with or without 1 μmol/L S1P for 2 days, and then were replaced with 10 μmol/L diaminofluorescein-2 diacetate solution for 30 minutes. After addition of 1 mmol/L L-arginine to the cells, fluorescence intensity was quantified using a computerized image analysis system with excitation and emission wavelengths of 490 nm and 540 nm, respectively, at room temperature. To minimize the effect of photobleaching, the observed fluorescence intensity in response to L-arginine was divided by the initial intensity prior to addition of L-arginine, and the ratio of fluorescence intensity was monitored during the experiment.

Western Blot Analyses.

Cultured SECs were washed with ice-cold physiological saline solution, harvested by scraping, and centrifuged at 1,500 rpm for 5 minutes. The cell pellet was lysed in a lysis buffer containing protease/phosphatase inhibitors as previously described.23 Equal amounts of cellular proteins were separated in 10% (Akt) and 7.5% (eNOS) SDS-PAGE, respectively, and transferred to polyvinylidene fluoride membranes. After blocking with 5% dry nonfat milk in Tris-buffered saline, membranes were incubated with appropriate primary antibodies such as anti–phospho-Akt (Ser-473, Cell Signaling Technology, Inc., MA) and anti–phospho-eNOS (Ser-1177, Cell Signaling Technology, Inc.) overnight at 4°C. Subsequently, secondary peroxidase-conjugated affinipure goat anti-Rabbit IgG antibodies (Jackson Immuno Research Laboratories, Inc., PA) were applied, and the specific bands were visualized using SuperSignal West Dura Extended Duration Substrate (Pierce Biotechnology, Inc., IL). Densitometric analyses of immunoblots were performed with NIH Image.Results were normalized by arbitrarily setting the densitometry of control cells to 1.0.

Electron Microscopy in Isolated Perfused Rat Liver.

To confirm the protective effect of S1P against ethanol-induced injury in SECs, rat liver tissues were investigated by electron microscopy. After perfusion of the liver from the portal vein with GBSS for 20 minutes, livers were perfused with (1) GBSS for 80 minutes (control); (2) GBSS for 20 minutes and subsequently 150 mmol/L ethanol for 60 minutes (ethanol); (3) 1 μmol/L S1P for 20 minutes and subsequently 1 μmol/L S1P plus 150 mmol/L ethanol for 60 minutes (S1P plus ethanol); (4) 1 μmol/L S1P plus 1 mmol/L NG-nitro-L-arginine methyl ester (L-NAME) for 20 minutes and subsequently 1 μmol/L S1P plus 1 mmol/L L-NAME plus 150 mmol/L ethanol for 60 minutes (S1P plus L-NAME plus ethanol); and (5) 1 μmol/L S1P alone for 80 minutes (S1P). A flow rate was maintained at 5 mL/min during the experiment. Liver tissues were fixed with 2.5% glutaraldehyde in 0.1 mol/L phosphate buffer for 2 hours and subsequently with 4% osmium tetroxide. Sections were cut on a Reichert ULTRACUT (Leica Microsystems), stained with uranyl acetate, and examined by transmission electron microscope (H-7100; Hitachi). Tissue sections were also observed with a scanning electron microscope (S-800; Hitachi).

Statistical Analysis.

Statistical analysis was performed using one-way ANOVA. A P value of less than .05 was considered statistically significant. All results are expressed as the mean ± SEM.

Results

Morphological Characterization of SECs in Primary Culture.

Under a phase-contrast microscope, the primary cultured SECs revealed a characteristic curved shape at day 2 in culture and became subconfluent at day 5 (Fig. 1A). Scanning electron microscopy showed a number of fenestrae in cells at day 2 in culture, confirming that characteristic properties of SECs are well preserved in these cells (Fig. 1B). The cultures were estimated to have greater than 95% purity, because fewer than 5% of the cells examined were devoid of fenestrae. All the experiments were performed in cells at day 2 in culture.

Figure 1.

Morphological characterization of SECs in primary culture. (A) Phase contrast micrographs of SECs during the time course in culture as described in Materials and Methods. SECs reveal their characteristic curved shape at day 2 and present subconfluent sheet at day 5. Representative photographs from six independent experiments are shown. (Original magnification ×100.) (B) Scanning electron micrographs show a number of fenestrae in sieve plate (arrows) at day 2 in culture. Representative photographs from three independent experiments are shown. (Original magnification ×5,000 [left panel], ×10,000 [right panel].) N, nucleus.

S1P Enhances DNA Synthesis of SECs in Primary Culture.

To test the effect of S1P on DNA synthesis in SECs, BrdU incorporation was examined. Incubation of cells with 1 μmol/L S1P for 2 days significantly increased BrdU labeling index. In contrast, in the cells treated with 150 mmol/L ethanol for 6 hours, BrdU labeling index was significantly decreased. Pretreatment of cells with S1P ameliorated this inhibitory effect of ethanol on DNA synthesis (Fig. 2). The effect of S1P on increased BrdU labeling index was cancelled by the simultaneous presence of 1 mmol/L L-NAME, a competitive NOS inhibitor, for 2 days (Fig. 2), suggesting that S1P-induced DNA synthesis is involved in NOS activation. In order to evaluate the results at the same condition as NO formation (see below), BrdU labeling index was investigated after treatment of cells with S1P for 2 days.

Figure 2.

Effect of S1P on DNA synthesis in SECs. As described in Materials and Methods, DNA synthesis of SECs was determined by BrdU incorporation, and the data were compared as a percentage of controls. The data were obtained from four independent experiments (S1P vs. control, P < .001; L-NAME/S1P vs. S1P, P < .001; ethanol vs. control, P < .001; S1P/ethanol vs. ethanol, P < .005; L-NAME/S1P/ethanol vs. S1P/ethanol, P < .001). The details of the experimental procedures are given in Results. BrdU, bromodeoxyuridine; S1P, sphingosine 1-phosphate; L-NAME, NG-nitro-L-arginine methyl ester.

S1P Protects SECs from Ethanol-Induced Apoptosis.

To determine whether ethanol induces apoptosis in SECs, TUNEL assay was performed (Fig. 3A-B). Ethanol-induced apoptosis was confirmed by transmission electron microscopy showing the condensation of nuclear chromatin and decreased cell size (Fig. 3C-D) and was demonstrated by a DNA ladder that illustrates DNA fragmentation (Fig. 4).

Figure 3.

Apoptosis of primary cultured rat SECs. Apoptotic cells were detected by TUNEL assay as described in Materials and Methods. (A) Control. (B) SECs were treated with 150 mmol/L ethanol for 6 hours. The arrows indicate TUNEL-positive cells. Representative photographs from six independent experiments are shown. (Original magnification ×100.) Apoptosis was confirmed by transmission electron microscopy. (C) Control. (D) Electron micrographs show the condensation of nuclear chromatin and decreased cell size (arrows) in ethanol-treated cells. Representative photographs from three independent experiments are shown. (Original magnification ×1,000.)

Figure 4.

DNA fragmentation in SECs treated with ethanol. Following incubation of cells with 150 mmol/L ethanol for 6 hours, electrophoresis shows DNA ladder that illustrates DNA fragmentation. Lane 1, positive control; lane 2, control; lane 3, ethanol; lane M, marker. Representative data from three independent experiments are shown.

Treatment of cells with 150 mmol/L ethanol for 6 hours induced a twofold increase in TUNEL-positive cells compared with controls; however, pretreatment of cells with 1 μmol/L S1P for 2 days significantly inhibited ethanol-induced apoptosis in SECs (Fig. 5). The inhibitory effect of S1P on ethanol-induced apoptosis was significantly attenuated by the simultaneous presence of 1 mmol/L L-NAME for 2 days, suggesting that S1P-induced inhibition of apoptosis is involved in NO synthase activation. To evaluate the results at the same condition as NO formation (see below), TUNEL assay was conducted after treatment of cells with S1P for 2 days.

Figure 5.

Effect of S1P on ethanol-induced apoptosis in SECs. Following incubation of cells with 1 μmol/L S1P for 2 days, cells were treated with 150 mmol/L ethanol for 6 hours, and then TUNEL-positive cells were assessed as a percentage of apoptotic cells. Data were obtained from four independent experiments (ethanol vs. control, P < .001; S1P/ethanol vs. ethanol, P < .001; L-NAME/S1P/ethanol vs. S1P/ethanol, P < .001). The details of the experimental procedures are given in Results. S1P, sphingosine 1-phosphate; L-NAME, NG-nitro-L-arginine methyl ester.

S1P Increases Intracellular Ca2+ Concentrations of SECs.

Addition of S1P to cells abruptly increased [Ca2+]i, which was sustained within observation (Supplementary Fig. 1A). However, the reduction of extracellular Ca2+ by 1 mmol/L egtazic acid decreased the duration of increase in [Ca2+]i (Supplementary Fig. 1B). These findings indicate that the increase in [Ca2+]i was partly due to mobilization from the internal sites of cells as well as influx of extracellular Ca2+ and that S1P acts as an extracellular ligand in intracellular calcium signaling. Furthermore, the presence of 150 mmol/L ethanol did not affect an S1P-elicited increase in [Ca2+]i (Supplementary Fig. 1C), suggesting that the biological effect of extracellular S1P is active in ethanol-induced damage of SECs.

S1P Induces NO Formation in SECs.

As described in Materials and Methods, NO was measured at a single-cell level via fluorescence microscopy in SECs. Addition of 1 mmol/L L-arginine, a substrate for eNOS, to media increased the ratio of fluorescence intensity in cells treated with S1P, but not in controls (Supplementary Fig. 2A), indicating that S1P induced NO formation in SECs. NO formation induced by S1P was abrogated by the simultaneous presence of 1 mmol/L L-NAME for 2 days (Supplementary Fig. 2B). Although eNOS activation peaked at 1 minute after stimulation of cells with S1P (see results shown below), a longer time incubation of cells with S1P was required to detect fluorescence intensity reflecting NO formation. This is presumably due to instability and low concentration of NO induced by eNOS as well as the methodological limitations of sensitivity to fluorescence intensity.

eNOS and Akt Activation by Western Blot Analysis.

Because NO is synthesized by eNOS in endothelial cells, Western blot analysis was performed to examine the effect of S1P on eNOS activity in SECs. As predicted, addition of 1 μmol/L S1P to cells induced eNOS phosphorylation on Serine-1177, which peaked at 1 minute after stimulation of cells with S1P without changing total eNOS levels (Fig. 6A). In contrast, phosphorylated Akt was not significantly induced by S1P (Fig. 6B), suggesting that S1P-induced NO production is not dependent of Akt signaling in primary cultured SECs. Following removal of VEGF from media, addition of 20 ng/mL VEGF to cells resulted in Akt phosphorylation (Fig. 6C), confirming that Akt phosphorylation is inducible in SECs. Furthermore, pretreatment of cells with LY294002—a specific inhibitor of PI-3K—for 60 minutes attenuated S1P-induced eNOS phosphorylation at 1 minute (Fig. 6D).

Figure 6.

Activation of eNOS and Akt in primary cultured SECs. Western blot analysis was performed as described in Materials and Methods. (A) Addition of 1 μmol/L S1P to cells induced eNOS phosphorylation. *P < .01 versus 0 minutes. (B) Addition of 1 μmol/L S1P to cells did not induce Akt phosphorylation. All of the points are not significantly different from 0 time. (C) Addition of 20 ng/mL VEGF to cells induced Akt phosphorylation. *P < .05 versus 0 minutes. **P < .01 versus 0 minutes. #P < .05 versus 0 minutes. (D) Pretreatment of cells with 10 μmol/L LY294002, an inhibitor of PI-3K, attenuated S1P-induced eNOS phosphorylation at 1 minute. *P < .001 versus control. **P < .05 versus S1P (+)/Ly294002 (−). (E) Pretreatment of cells with 10 μmol/L trifluoriperazine, a CaM inhibitor, inhibited S1P-induced eNOS phosphorylation at 1 minute. *P < .01 versus control. **P < .001 versus S1P (+)/trifluoriperazine (−). Data were obtained from at least four independent experiments. eNOS, endothelial NO synthase; S1P, sphingosine 1-phosphate; VEGF, vascular endothelial growth factor; TFP, trifluoriperazine.

Inhibition of Calmodulin Attenuates S1P-Induced eNOS Activity in SECs.

To test whether S1P-induced Ca2+ increase is involved in eNOS activation and subsequent NO formation, eNOS phosphorylation was examined by Western blot analysis after incubation of cells with 10 μmol/L trifluoriperazine, an inhibitor of calmodulin, for 30 minutes. Consequently, trifluoriperazine significantly inhibited S1P-induced eNOS phosphorylation at 1 minute, suggesting that S1P-induced NO production is mediated by increased activity of Ca2+-sensitive eNOS (Fig. 6E).

S1P Protects SECs Against Ethanol-Induced Injury in Isolated Perfused Rat Liver.

To confirm the protective effect of S1P against ethanol-induced injury in SECs, liver tissues obtained from isolated perfused rat liver were investigated in electron microscopy. In controls, characteristic sinusoids lined with a normal elongated endothelial cell were seen (Fig. 7A,a; Fig. 8A,a). In ethanol-perfused liver, endothelial injury was apparent with less abundant cytoplasm and nuclear abnormalities such as crenellation and chromatin condensation (Fig. 7B,b; Fig. 8B,b). However, S1P inhibited these morphological changes caused by ethanol (Fig. 7C,c; Fig. 8C,c), which was cancelled by the simultaneous presence of 1 mmol/L L-NAME (Fig. 7D,d; Fig. 8D,d). This suggests that the protective effect of S1P against ethanol-induced injury in SECs is at least in part involved in NOS activation in isolated perfused rat liver. The liver tissue perfused with S1P alone was similar to controls (Fig. 7Ee; Fig. 8E,e). These results were quantified by counting injured SECs, which were characterized by nuclear abnormalities, in transmission electron microscopy (Table 1). The data were obtained from three independent experiments.

Figure 7.

Transmission electron micrographs of in situ isolated perfused rat liver tissues. Experiments were performed as described in Materials and Methods. (A,a) In controls, characteristic sinusoids lined with normal elongated endothelial cells were seen. (B,b) In ethanol-perfused liver, endothelial injury was apparent with less abundant cytoplasm and nuclear abnormalities such as crenellation and chromatin condensation. (C,c) Preperfusion of liver with S1P inhibited these morphological changes caused by ethanol. (D,d) Preperfusion of liver with S1P in the simultaneous presence of 1 mmol/L L-NAME cancelled the inhibitory effect of S1P on ethanol-induced morphological changes. (E,e) The liver tissue perfused with S1P alone was similar to controls. Representative photographs from three independent experiments are shown. (Original magnification ×1,000 [A-E], ×3,000 [a-e].) S, sinusoid; K, Kupffer cell; H, hepatocyte; E, endothelial cell.

Figure 8.

Scanning electron micrographs of in situ isolated perfused rat liver tissues. The experimental procedure is identical to that in Fig. 6. Representative photographs from three independent experiments are shown. (Original magnification ×2,000 [A-E], ×10,000 [a-e].) E, endothelial cell; H, hepatocyte; S, sinusoid.

Table 1. Protective Effect of S1P on SECs in Isolated Perfused Rat Liver Tissues (% of Control)
 ControlEthanolS1P/EthanolL-NAME/S1P/EthanolS1P
  • NOTE. Results are expressed as the mean ± SEM for 8-10 visual fields per sample in three independent experiments.

  • *

    P < .001 versus control.

  • **

    P < .001 versus ethanol.

  • P < .001 versus S1P/ethanol.

Injured cells100.0 ± 10.7352.7 ± 25.0*202.7 ± 15.7**339.0 ± 24.7101.7 ± 28.8

Discussion

This study showed that S1P protects SECs from ethanol-induced injury in primary culture and isolated perfused liver in rats, and that the protective effect of S1P may be mediated by activation of Ca2+-sensitive eNOS and subsequent NO formation. S1P also ameliorated the decreased DNA synthesis of cells caused by ethanol. We thus propose that S1P, which is possibly released from activated platelets in response to ethanol consumption, could contribute to sinusoidal protection and remodeling in alcoholic liver injury.

The mechanism of alcoholic liver disease has been largely investigated in parenchymal hepatocytes. In hepatocytes, long-term exposure to ethanol is essential for its complex metabolism leading to cellular injury and dysfunction, while it is unlikely that ethanol is metabolized in SECs. In contrast, because SECs are directly exposed to high concentrations of ethanol rather than hepatocytes, it may be possible that ethanol-induced injury in SECs plays an important role in the progression of liver damage through the disturbance of integrity of endothelium that acts as a barrier. However, little is known about the mechanisms underlying the protection of SECs against ethanol-induced injury.

Recently, S1P has been revealed to mediate numerous biological effects, including cell proliferation,15 wound healing,17 and inhibition of apoptosis,18, 19 and to regulate vascular maturation and angiogenic processes in endothelial cells.16, 28 S1P was originally considered to be formed merely as an intermediate in the detoxification of sphingosine by its phosphorylation and subsequent degradation. The question of how such a simple molecule can have diverse roles has been resolved by the discovery that it belongs to a class of lipid mediators that function not only inside cells but also as ligands for specific cell-surface receptors (e.g., S1P receptors).20

In the liver, it has been indicated that S1P may play a role in wound healing through increased proliferation and contraction of stellate cells in vitro,28 and that it exerts an inhibitory effect on growth factor–induced DNA synthesis in hepatocytes.29 However, the effect of S1P on SECs has remained unclear in alcoholic liver disease, in which platelets appear to play an important role in sinusoidal microcirculation. Platelets have a highly active form of sphingosine kinase that rapidly converts sphingosine into S1P and have a relative deficiency of sphingosine lyase, which is the enzyme responsible for S1P catabolism and breakdown.30 Consequently, platelets store large amounts of S1P and are the cellular component that is primarily responsible for the concentration of S1P found in plasma.13

Platelets accumulate in the liver after LPS administration in vivo,21 and thrombin that is essential for LPS-induced liver injury is the most potent stimulator of platelets.14 Given the accumulating evidence that gut-derived LPS plays a critical role in alcoholic liver disease,31, 32 it is likely that S1P released from activated platelets in response to ethanol consumption plays a role in the process of sinusoidal endothelial injury.

In this study, we found that extracellular S1P protected SECs from ethanol-induced injury in primary culture and isolated perfused liver in rats. Recent evidence suggests that S1P promotes survival in macrophages,33 Schwann cells,34 T lymphocytes,35 and fibroblasts.36 S1P also protects endothelial cells from apoptosis induced by C2-ceramide, tumor necrosis factor α, and anti-Fas antibody.18, 37, 38 However, the mechanism underlying antiapoptotic effect of S1P is complex and is not fully understood.

It has been suggested that S1P activates Akt, which is considered to be a downstream mediator of PI-3K–dependent survival signaling.39 Akt subsequently increases eNOS activity by phosphorylation at Ser-1177 and Ser-1179 for human and bovine eNOS, respectively, leading to elevation of NO production,40, 41 which is an important downstream effector in antiapoptotic signaling.42 NO has been suggested to play an important regulatory role in apoptotic cell death.43 High or pathological concentrations of NO produced from inducible NO synthase induce apoptosis, whereas low concentrations of NO produced from eNOS or pharmacological concentrations of exogenous NO released by NO donors reduce apoptosis.42

In our study, however, S1P-induced activation of Akt, as judged by phosphorylation at the Ser-473 moiety, was not detected, whereas VEGF resulted in Akt phosphorylation in SECs, confirming that Akt phosphorylation is inducible in these cells. A likely explanation is that cell-type specificity exists in the antiapoptotic signaling pathways activated by phospholipids.44 Our results suggest that PI-3K is at least in part involved in eNOS activation, because the specific PI-3K inhibitor LY294002 attenuated eNOS activity. Recent studies have reported that in osteoblastic cells, the antiapoptotic effect of S1P is PI-3K–dependent, whereas Akt activation is not induced by S1P.44 The mechanisms underlying cell-type specificity have not been elucidated; however, it cannot be excluded that the differences of S1P receptors in cell-type specificity are involved in the diverse downstream signaling, which may partly explain our results.

Alternatively, eNOS is a highly regulated Ca2+/calmodulin (CaM)-dependent enzyme that plays a key role in many signaling cascades by catalyzing the conversion of L-arginine and oxygen to L-citrulline and the labile gas NO.45, 46 CaM is a ubiquitously expressed and highly conserved acidic protein that mediates a broad range of intracellular calcium-regulated enzymes.47, 48 It has been well documented that although inducible NO synthase is able to bind CaM with extremely high affinity even at low [Ca2+]i, increase in [Ca2+]i is required for the binding of eNOS to CaM leading to full activation of eNOS.46 Our results revealed that S1P increased intracellular Ca2+ concentrations in SECs, and inhibition of CaM attenuated S1P-induced eNOS activity in SECs, suggesting the importance of Ca2+/CaM complex in eNOS activation resulting in NO formation. Collectively, our results indicate that S1P caused eNOS phosphorylation and subsequent NO formation in SECs in primary culture, thus leading to its antiapoptotic effect largely mediated by NO signaling pathways. This idea is further supported by the result that protective effect of S1P on ethanol-induced apoptosis was cancelled by L-NAME, a NO synthase inhibitor.

In conclusion, we provide evidence that S1P contributes to sinusoidal protection against ethanol-induced injury through Ca2+-sensitive eNOS activation and subsequent NO formation. S1P-induced proliferation of SECs may cooperatively act in the regulation of sinusoidal restoration in ethanol-induced liver injury. Although further research is required to elucidate the molecular interaction between Ca2+, CaM and eNOS activity, our results suggest a possible mechanism for the regulation of ethanol-induced apoptosis in SECs by S1P. The role of S1P in normal physiology is uncertain. However, our findings may lead to a better understanding of S1P as a target for novel therapeutic strategies in alcoholic liver injury.

Ancillary