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International Journal of Experimental Pathology logoLink to International Journal of Experimental Pathology
. 2008 Aug;89(4):241–250. doi: 10.1111/j.1365-2613.2008.00590.x

Endothelial nitric oxide synthase is a critical factor in experimental liver fibrosis

Tung-Ming Leung *,†,1, George L Tipoe †,1, Emily C Liong †, Thomas YH Lau ‡, Man-Lung Fung §, Amin A Nanji *
PMCID: PMC2525781  PMID: 18429990

Abstract

Reduced expression of endothelial nitric oxide synthase (eNOS) in chronic liver disease can reduce hepatic perfusion and accelerate fibrosis. The relationship between eNOS expression and liver fibrogenesis remains unclear. We investigated whether l-arginine attenuated chronic liver fibrosis through eNOS expression. Chronic liver injury was induced by administration of carbon tetrachloride (CCl4) to mice for 8 weeks. 5-Methylisothiourea hemisulphate (SMT), an iNOS inhibitor, or l-arginine, a NOS substrate were injected subcutaneously. CCl4-induced hepatotoxicity, oxidative stress and accumulation of collagen were detected in the liver. The expression levels of inducible NOS (iNOS) and nuclear factor kappa-B (NF-κB) activity in the liver after CCl4 treatment were increased but eNOS expression and activator protein-1 (AP-1) activity were decreased. Both SMT and l-arginine effectively reduced CCl4 induced oxidative stress and collagen formation, but l-arginine showed a significantly greater suppression of collagen formation, iNOS expression and NF-κB activity. l-Arginine also restored the level of eNOS and AP-1 activity. l-Arginine was more effective than SMT in suppressing liver fibrosis. l-Arginine might improve NO production which facilitates hepatic blood flow and thus retards liver fibrogenesis. Our results showed that the reduced eNOS expression in CCl4-treated mice was reversed by l-arginine. Furthermore, l-arginine also reversed the reduced AP-1 activity, an eNOS promoter.

Keywords: carbon tetrachloride, chronic liver injury, l-arginine, nitric oxide synthase

Regardless of aetiology, liver injury increases a variety of cytokines and growth factors which lead to liver fibrosis which is observed in many chronic liver diseases. Accumulation of extracellular matrix (ECM) in the liver can lead to cirrhosis or hepatocellular carcinoma (Milani et al. 1995; Bissell 1998; Giannelli et al. 2003; Bataller & Brenner 2005). It is important to attenuate the process of ECM accumulation in the liver by identifying the factors that cause chronic liver injury and fibrosis.

One of the common complications in advanced liver diseases is portal hypertension (Gines et al. 2004). The mechanisms that lead to an increase in intra-hepatic resistance are not completely understood but are thought to involve a change in expression/activity of nitric oxide synthases (NOS) and the production of nitric oxide (NO). NO is produced predominantly through the enzymatic action of inducible (iNOS) and endothelial (eNOS) nitric oxide synthases in the liver (Marletta et al. 1998). NO has versatile functions in the body which include cell signalling as a second messenger, antimicrobial ability and is a very potent vasodilator in the regulation of vascular tone (Mittal et al. 1994; Gupta et al. 1998; Laroux et al. 2001; Blaise et al. 2005). eNOS-derived NO in the liver is essential in the regulation of normal hepatic blood flow (Palmer et al. 1987). l-Arginine is a substrate for all isoforms of NOS for the production of NO (Bruckdorfer 2005), and has been shown to be effective in the relief of portal hypertension in patients with liver cirrhosis (Kakumitsu et al. 1998).

Other work has indicated that there is a re-distribution of the expression of both eNOS and iNOS in chronic liver disease (Wei et al. 2002). Furthermore, the expression of both eNOS and iNOS is diminished in sinusoidal endothelial cells isolated from liver exposed to prolonged insults (Petermann et al. 1999). These studies provide evidence that different isoforms of NOS and the production of NO play different roles in the liver. Thus, altered expression of these different isoforms of NOS is a possible key factor in the progression of chronic liver injury.

We hypothesized that eNOS-derived NO was a major factor in accelerating ECM accumulation by altering the expression of various pro-fibrogenic factors induced in chronic liver injury. The aim of this study was therefore, together with examination of various fibrogenic factors, to determine the roles of two of the major isoforms of NOS (eNOS and iNOS) in response to arginine and an iNOS inhibitor. We used a chronic liver fibrosis animal model to determine the effect of arginine and the iNOS inhibitor on the different isoforms of NOS and progression of liver fibrosis.

Materials and methods

Animal model and treatments

Eight-week-old male ICR mice were maintained under standard condition with free access to water and chow in compliance with the requirements of the University of Hong Kong and the National Institute of Health guidelines. Mice were divided into eight groups (n = 10–15): (1) vehicle only (normal saline/olive oil); (2) carbon tetrachloride (CCl4; 50 μl/kg); (3) d-arginine (200 mg/kg); (4) d-arginine + CCl4; (5) 5-methylisothiourea hemisulphate (SMT, 10 mg/kg); (6) l-arginine (200 mg/kg); (7) SMT + CCl4; (8) l-arginine + CCl4 (d-arginine, l-arginine and SMT were purchased from Sigma, St. Louis, MO, USA). CCl4 was dissolved in olive oil and injected twice a week intraperitoneally. SMT, l-arginine and d-arginine were dissolved in normal saline and injected subcutaneously daily and given for 30 min prior to CCl4 treatment. The mice were killed after the 8-week treatment. Mice from all experimental groups were fasted for 12 h before killing.

Tissue processing and Sirius Red staining

Fresh liver tissue blocks were fixed in 4% phosphate-buffered formalin and dehydrated. Paraffin-embedded tissues were cut into 5 μm sections. Quantitative analysis of collagen accumulation in the tissues was visualized by Sirius Red staining and morphometric analysis. Briefly, the tissue sections were stained with 0.1% picro-Sirius Red (Polysciences Inc., Washington, DC, USA) in saturated aqueous picric acid for 1 h followed by differentiation in 0.01% hydrochloric acid. The Sirius Red-stained collagen was classified into total collagen, collagen around the central veins and pericellular fibrosis. The Sirius Red-stained collagen was quantified using LEICA Qwin Image Analyzer (Leica Microsystems Ltd, Milton Keynes, UK). All sections were examined by the same person.

Measurement of serum alanine aminotransferase (ALT) and total 8-isoprostane

Serum ALT and 8-isoprostane were performed as described previously (Tipoe et al. 2006). The level of serum ALT indicated the degree of necrosis and the total serum 8-isoprostane, a measurement of oxidative stress was measured by the 8-Isoprostane EIA Kit (Cayman Chemical, Ann Arbor, MI, USA).

Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis

Total RNA was extracted from liver tissues using the NucleoSpin Nucleic Acid Purification Kits (CLONTECH Laboratories, Inc., Palo Alto, CA, USA). First-strand cDNA was prepared by following the instructions in SuperScriptTM First-Strand Synthesis System for RT-PCR kit (Life Technologies, Carlsbad, CA, USA). Forward and reverse primers used in PCR are shown in Table 1. The thermal cycles of PCR are as follows: denaturing of the PCR mixture at 95 °C for 15 min and then amplification (95 °C for 1 min, 60 °C for 1 min, 72 °C for 1 min) for 30 cycles [transforming growth factor-β1 (TGF-β1) and procollagen-I]; 35 cycles (eNOS) and 37 cycles (iNOS). A final extension was carried out at 72 °C for 10 min. The PCR products were resolved in 2% agarose gel.

Table 1.

Primers used for RT-PCR

Target gene Sequence
iNOS
 Forward 5′-GTGGTGACAAGCACATTTGG-3′
 Reverse 5′-GGCTGGACTTTTCACTCTGC-3′
eNOS
 Forward 5′-GACCCTCACCGCTACAACAT-3′
 Reverse 5′-CACAGAAGTGGGGGTATGCT-3′
TGF-β1
 Forward 5′-CTTCAGCTCCACAGAGAAGAACTGC-3′
 Reverse 5′-CACGATCATGTTGGACAACTGCTCC-3′
Procollagen-I
 Forward 5′-TGCCGTGACCTCAAGATGTGCC-3′
 Reverse 5′-CATCCACAAGCGTGCTGTAGGTG-3′
GAPDH
 Forward 5′-CCTTCATTGACCTCAACTACATGGT-3′
 Reverse 5′-TCATTGTCATACCAGGAAATGAGCT-3′
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RT-PCR, reverse transcriptase-polymerase chain reaction; iNOS, inducible nitric oxide synthase; eNOS, endothelial NOS; TGF-β1, transforming growth factor-β1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Western blot analysis

Cytosolic and nuclear protein were extracted as described previously (Tipoe et al. 2006). Cytosolic protein was mixed with sample buffer (0.1 m Tris-HCl pH 6.8, 20% glycerol, 4% SDS, 0.2% bromophenol blue and 5.25%β-mercaptoethanol) followed by electrophoresis in polyacrylamide gel. The protein was transferred to Immun-BlotTM PVDF Membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA) in a TE series transfer electrophoresis unit. The membrane was incubated in blocking buffer for 3 h before incubation with primary antibody overnight at 4 °C: iNOS (1:500 dilution; Transduction Lab, San Jose, CA, USA); eNOS (1:1000 dilution; Transduction Lab); Nitrotyrosine (1:1000 dilution; Zymed Laboratories Inc., CA, USA); β-actin (Sigma). The membrane was incubated in secondary antibody (1:2000 dilution) for 2 h at room temperature. The expression of the antibody linked protein was detected by an ECL Western Blotting detection kit (Amersham Pharmacia Biotech Inc., Buckinghamshire, UK).

Electrophoretic-mobility shift assay (EMSA)

Nuclear factor-kappa B (NF-κB) and activator protein-1 (AP-1) are the major transcription factors for iNOS and eNOS respectively. Briefly, the phosphorylated and purified consensus NF-κB or AP-1 oligonucleotides (Promega, Madison, WI, USA) was mixed with 24 μg of nuclear protein extract and 10× Gel Shift Binding Buffer (200 mm Tris-HCl pH 7.8, 1 m NaCl, 50 mm MgCl2, 10 mm EDTA and 50 mm dithiothreitol). The mixture was incubated at room temperature for 20 min before loading onto a 4% non-denaturing polyacrylamide gel. The gel was dried and exposed to an X-ray film and the signal was quantified using laser scanning densitometry. As in previous studies, specificity of NF-κB binding was confirmed by competition assays and the ability of a specific antibody to supershift protein–DNA complexes. In the competition assay, the addition of 100-fold excess of unlabelled competitor consensus oligonucleotide prevented binding. Supershift experiments confirmed the presence of the p50 subunit in the binding complexes.

Statistical analysis

Results are expressed as mean ± standard error of mean (SEM). Comparisons among different groups were performed using the Mann–Whitney U-test (two-tailed) using GraphPad Prism (GraphPad Software, Inc., San Diego CA, USA). A P-value of <0.05 was regarded as statistically significant.

Results

Role of l-arginine and SMT in the modulation of CCl4-induced liver injury and oxidative stress

Increased activity of serum ALT was observed after chronic CCl4 intoxication (P < 0.01) (Figure 1a). Both l-arginine and SMT significantly reduced the CCl4-induced increase in the ALT level (P < 0.01). In CCl4-induced liver injury, the serum level of 8-isoprostane and the formation of nitrotyrosine in liver were significantly increased when compared with controls (P < 0.05) (Figure 1b,c). The administration of l-arginine and SMT significantly reduced the serum levels of total 8-isoprostane and nitrotyrosine (P < 0.05) when compared with the CCl4 and d-arginine + CCl4 groups.

Figure 1.

Figure 1

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(a) Serum activity of alanine aminotransferase (ALT). The groups treated with carbon tetrachloride (CCl4) and d-arginine + CCl4 showed the highest level of serum ALT indicating liver damage after chronic administration of CCl4 (*P < 0.01 compared with control). Pre-treatment with SMT or l-arginine followed by CCl4 showed a reduced ALT level (**P < 0.01 compared with CCl4 the treatment group). The oxidative stress markers serum total 8-isoprostane (b) and nitrotyrosine (c) show the highest level in the CCl4 and d-arginine + CCl4 groups (*P < 0.05, and †P < 0.001, respectively, compared with control). Pre-treatment with SMT or l-arginine significantly reduced the total 8-isoprostane and nitrotyrosine levels in comparison with the CCl4 group (**P < 0.05).

Administration of l-arginine showed a greater degree of suppression than SMT in the accumulation of collagen

The result of Sirius Red staining in the livers of the experimental groups is shown in Figure 2. Qualitatively, only minor fibrotic changes such as fibrosis around the central vein and pericellular fibrosis were present in normal liver (Figure 2a). In contrast, numerous sites of collagen deposition were observed in CCl4-treated mice. Major sites of collagen accumulation were concentrated around the central vein (Figure 2b). The treatment with either l-arginine or SMT effectively reduced the amount of collagen deposition (Figure 2c,d). Calculated data revealed that the degree of Sirius Red staining was significantly reduced in the SMT + CCl4 and l-arginine + CCl4 groups (Figure 2e–g).

Figure 2.

Figure 2

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Quantitative analysis of collagen in the various treatment groups. Sirius Red staining of liver sections in (a) normal control, (b) carbon tetrachloride (CCl4)-treated rat, treatment with (c) 5-methylisothiourea hemisulphate (SMT) and CCl4 and (d) l-arginine and CCl4. The collagen is stained in red. Quantitative analysis of the amount of collagen deposited in the liver sections showing the amount of collagen from the (e) pericellular area, (f) central vein and (g) total amount of collagen in the sections. CCl4 and d-arginine + CCl4 groups show a significant increase in the amount of collagen accumulated compared with control groups (†P < 0.001 and *P < 0.05 respectively). Groups pre-treated with either SMT or l-arginine significantly reduced CCl4-induced collagen deposition (‡P < 0.001 compared with the CCl4 treatment group). Pre-treatment with l-arginine showed a stronger inhibitory effect than that of SMT (**P < 0.001) (magnification ×200).

A greater than 10-fold increase in the amount of collagen was detected in the pericellular area (P < 0.001) and a two- to threefold increase seen in the central vein (P < 0.001) and total amount of collagen (P < 0.001), respectively, in the CCl4 and d-arginine + CCl4 groups compared with controls (Figure 2b–d). A significant reduction in collagen deposition was detected in the groups pre-treated with SMT and l-arginine. Of note is that the l-arginine + CCl4 group showed a greater reduction in the amount of collagen than the SMT + CCl4 group (P < 0.01).

Effect of l-arginine and SMT on mRNA levels of TGF-β1 and procollagen-I

Semi-quantitative RT-PCR for TGF-β1 and procollagen-I mRNAs was performed to further evaluate hepatic fibrosis. In CCl4-treated rats, (CCl4 and d-arginine + CCl4 groups), the mRNA levels of TGF-β1 and procollagen-I were markedly increased vs. controls (P < 0.001) (Figure 3a,b). The CCl4-induced expression of TGF-β1 and procollagen-I was significantly reduced in the l-arginine and SMT groups (P < 0.001). The results of TGF-β1 and procollagen-I mRNAs are consistent with the results obtained with Sirius Red-stained collagen formation (Figure 2). Importantly, l-arginine treatment exhibited a greater suppression in the expression of mRNAs of both TGF-β1 and procollagen-I than in the SMT-treated group (P < 0.001).

Figure 3.

Figure 3

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Semi-quantitative PCR showing the mRNA levels of (a) transforming growth factor-β1 (TGF-β1) and (b) procollagen-I. Treatment with carbon tetrachloride (CCl4) and d-arginine + CCl4 showed a significantly increased level of expression of TGF-β1 (†P < 0.001 compared with control). 5-Methylisothiourea hemisulphate (SMT) + CCl4 and l-arginine + CCl4 show significant reduction in the expression TGF-β1 induced by CCl4 (‡P < 0.001 compared with the CCl4 treatment group), with l-arginine showing a greater reduction than SMT in inhibiting the expression of TGF-β1 (**P < 0.001). The expression of procollagen-I is shown to be increased in the groups treated with CCl4 and d-arginine + CCl4 (†P < 0.001 compared with control). Pre-treatment with l-arginine or SMT showed a significant reduction in CCl4-induced expression of procollagen-I (‡P < 0.001 compared with the CCl4 treatment group). Pre-treatment with l-arginine showed a stronger inhibitory effect than SMT (**P < 0.001).

DNA-binding activity of NF-κB and iNOS expression in CCl4-induced fibrosis

The DNA-binding activity of NF-κB and the expression of the NF-κB-inducible isoform of NOS are shown in Figure 4. A fourfold increase in NF-κB binding activity was detected in CCl4 and d-arginine + CCl4 groups compared with controls (P < 0.01) (Figure 4a). In the groups pre-treatment with l-arginine or SMT, the activity of NF-κB was significantly reduced to about half of the level seen in the CCl4 and D-arginine + CCl4 groups (P < 0.001). Consistent with its decreased activity, the NF-κB-regulated gene, iNOS, l-arginine showed a suppression of mRNA and protein levels similar to that of SMT (P < 0.05 and P < 0.001 respectively). iNOS showed an approximately twofold increase in both mRNA and protein levels after the treatment with CCl4 or d-arginine + CCl4 (P < 0.001 and P < 0.05 respectively) when compared with controls (Figure 4b,c). Pre-treatment with l-arginine or SMT showed a significant and marked reduction in iNOS mRNA and protein expression (P < 0.05 and P < 0.001 respectively). l-arginine showed a greater reduction than SMT in the levels of iNOS mRNA and protein (P < 0.05).

Figure 4.

Figure 4

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(a) DNA binding activity of nuclear factor kappa-B (NF-κB) in various experimental groups. In the group treated with carbon tetrachloride (CCl4) and d-arginine + CCl4, increased DNA-binding activity of NF-κB was seen (†P < 0.01 compared with control). Pre-treatment with l-arginine or SMT showed a significant reduction in the effect of CCl4-induced activity of NF-κB (‡P < 0.001). To evaluate the NF-κB-regulated gene, iNOS, expression of iNOS (b) mRNA and (c) protein was measured. In CCl4-induced liver injury and the group treated with d-arginine + CCl4, a high level of iNOS mRNA and protein were observed (†P < 0.001 and *P < 0.05, respectively, vs. control). Pre-treatment with SMT and l-arginine showed a significant inhibitory effect on the expression of iNOS mRNA and protein levels (‡P < 0.001 and **P < 0.05, respectively, compared with the CCl4 treatment group).

Decreased DNA-binding activity of AP-1 and eNOS expression in CCl4-treated mice

The DNA-binding activity of AP-1 and the expression level of eNOS are shown in Figure 5. In contrast to the increase in NF-κB, the activity of AP-1 was significantly reduced in CCl4 and d-arginine + CCl4 groups (P < 0.01) (Figure 5a). The effect of CCl4-induced downregulation of AP-1 was abrogated in the l-arginine and SMT-treated groups (P < 0.001). Similar to the trend seen with AP-1 activity, the levels of eNOS mRNA and protein were also significantly reduced, after CCl4-treatment, to about half of the level seen in the CCl4 group (P < 0.01) (Figure 5b,c). Furthermore, pre-treatment with l-arginine or SMT significantly increased the eNOS mRNA (P < 0.05) and protein levels (P < 0.05). Furthermore, the eNOS levels returned to the levels seen in the control groups.

Figure 5.

Figure 5

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(a) DNA binding activity of activator protein-1 (AP-1) in various experimental groups. AP-1 activity was significantly decreased in the groups treated with carbon tetrachloride (CCl4) and d-arginine + CCl4 (†P < 0.01 compared with control). Pre-treatment with l-arginine or SMT showed the restoration of AP-1 activity to a level similar to that observed in the control groups (‡P < 0.001 when compared with the CCl4 treatment groups). The expression levels of endothelial nitric oxide synthase (eNOS) (b) mRNA and (c) protein are also shown to be significantly decreased in the CCl4 and d-arginine + CCl4vs. control (*P < 0.01). In the groups pre-treated with SMT or l-arginine, the level of expression of eNOS was similar to that seen in controls (**P < 0.05 compared with the CCl4 treatment group) and the restoration of eNOS mRNA and protein paralleled the similar change in AP-1 binding seen in the CCl4-treated and l-arginine and SMT groups.

Discussion

Distinctive pathways are mediated by eNOS and iNOS in CCl4-induced fibrotic liver injury

As a prototypical endothelium relaxing factor, NO is an important determinant of blood vessel tone and hepatic blood supply. The recognition that all three isoforms of NOS are expressed in liver has led to several questions regarding the role of NO in liver. For example, whether NO is beneficial or detrimental in chronic liver diseases is still inconclusive. It is thought that the increased NO production in liver cirrhosis is a result of the increased incidence of endotoxaemia and the subsequent increase in cytokine production (Vallance & Moncada 1991). This increase in NO production is suggested to be responsible for the hyperdynamic circulation in liver cirrhosis (Vallance & Moncada 1991). The damaging effect of NO is supported by the finding that a high level of plasma nitrites and nitrates is correlated with severe damage in the liver (Moussa et al. 2000; Coskun et al. 2001). However, other studies have shown contradictory findings which show that increasing the availability of NO by various NO donors was protective (Bhathal & Grossman 1985; Garcia-Pagan et al. 1999; Fiorucci et al. 2001).

The current study showed that the expression of eNOS and iNOS was altered differently in chronic liver injury. In the model of CCl4-induced liver fibrosis, eNOS expression was significantly reduced and was partly attributed to reduced DNA-binding activity of AP-1. In contrast, the expression of iNOS and the DNA-binding activity of its transcription factor, NF-κB, were increased. Therefore, the adverse effects contributed by NO in chronic liver injury might be due to increased iNOS or reduced eNOS or both. In the group treated with the iNOS inhibitor, SMT, a low level of NO production might be expected because of the reduction in iNOS activity (Figure 4). Treatment with the NOS substrate, l-arginine, led to increased levels of AP-1 and eNOS in comparison with CCl4-treated mice. Of note, treatment with l-arginine decreased but did not enhance the process of fibrosis, revealing the potential dichotomy between regulation of NOS isoforms in the progression of fibrosis. Both SMT and l-arginine reduced the level of the marker of oxidative stress, serum 8-isoprostane and the formation of nitrotyrosine, to the basal level. Treatment with l-arginine was not only more effective than SMT in reducing levels of proinflammatory mediators but also decreased the severity of CCl4-induced liver fibrosis.

Adverse effect of increased expression of iNOS in chronic liver injury

Our results show that the expression level of iNOS was increased after chronic administration of CCl4 (Figure 4b,c). The high level of NO production secondary to iNOS induction is one of the major hallmarks of the inflammatory response (Moncada 1999). A high concentration of NO produced by iNOS in response to various stimuli such as cellular injury might induce oxidative stress by initiating lipid peroxidation and the formation of peroxynitrite (ONOO−). The treatment with the iNOS inhibitor, SMT, effectively reduced the expression level of iNOS as well as the degree of collagen deposition in the liver, suggesting that the protective effect of SMT in chronic liver injury might occur through the attenuation of inflammatory response.

Altered eNOS expression in liver injury is an important factor in the progression of liver fibrogenesis

Despite the fact that l-arginine and SMT have similar effects in mitigating CCl4-induced liver fibrosis, l-arginine was clearly more potent in attenuating the accumulation of collagen. Consistent with observations in the present study, Milani et al. (1992) also show increased expression levels of TGF-β1 and procollagen-I in chronic liver injury. Procollagen-I is the precursor of various types of collagen synthesized for the restructuring process (Canty & Kadler 2005). TGF-β1 is known to promote the process of liver regeneration after damage (Jung et al. 2000; Marek et al. 2002). TGF-β1 is also responsible for the stimulation of ECM synthesis and inhibits degradation of the excess ECM in the liver tissue. The expression of eNOS and the DNA-binding activity of the transcription factor AP-1 were reduced in CCl4-treated mice (Figure 5). AP-1 is a major transcription factor that regulates the transcription of eNOS (Hoffmann et al. 2001), and its decreased activity might, in part, explain the decrease in eNOS mRNA and protein levels. Previous studies have shown impaired NO production in the sinusoidal areas in cirrhotic liver (Loureiro-Silva et al. 2003). This results in endothelial dysfunctions in cirrhotic livers (Gupta et al. 1998; Shah et al. 1999; Cahill et al. 2001). Although the expression level of eNOS protein was not altered in cirrhotic liver or chronic liver diseases when compared with normal controls (Shah et al. 1999; Leifeld et al. 2002; Wei et al. 2002), a re-distribution of the expression of eNOS has been observed in liver cirrhosis (Wei et al. 2002). Wei et al. (2002)demonstrated a high level of eNOS expression in sinusoidal endothelial cells in the livers of sham rats in contrast to a markedly reduced expression of eNOS in the sinusoidal endothelial cells in the cirrhotic livers of bile duct-ligated rats. The reduction in eNOS expression in the sinusoidal endothelium might lead to decreased eNOS-derived NO production and subsequently result in increased resistance in the hepatic microcirculation. Although measurements of NO would have been potentially helpful in the present study, we did not isolate the individual cell types (endothelial cells, Kupffer cell, hepatic stellate cells and hepatocytes) to clearly identify the sources of NO production. Measurement of NO in whole liver would have produced a result of the complex interplay among the different hepatic cell types which would make it difficult to interpret.

Recent studies have also revealed the importance of the renin–angiotensin system (RAS) in the progression of liver fibrosis. The RAS is a hormonal system which regulates the blood pressure in the body. It has also been shown that angiotensin-II, a key vasoconstrictor in the RAS, is actively involved in fibrogenesis (Yoshiji et al. 2006; Li et al. 2007). The plasma level of angiotensin-II, a key vasoconstrictor polypeptide, is significantly increased in patients with liver cirrhosis and is found to be a key factor in inducing portal hypertension (Ballet et al. 1988; Garcia-Pagan et al. 1995). Furthermore, angiotensin-II also induces the proliferation of hepatic stellate cells, increases the mRNA level of TGF-β1 and collagen-I, and stimulates the formation of ECM (Bataller et al. 1998, 2000; Marshall et al. 2000). The effectiveness of l-arginine in maintaining the normal production of eNOS-derived NO level might help in keeping the balance between the vasodilator and vasoconstrictor and thus attenuate the progression of fibrosis.

Role of l-arginine in decreasing hepatic fibrosis: possible mechanisms

Impaired endothelial function is an early marker of many diseases including chronic liver disease and is often present before structural alterations in sinusoidal cells are detectable. The observation that supplementation of the NO precursor l-arginine prevented fibrosis in CCl4-treated mice provides support for the potential therapeutic value of manipulation of the arginine/NO pathway.

Although NO synthase is theoretically saturated with its natural substrate, exogenous administration of l-arginine leads to restoration of impaired endothelial function and the phenomenon is known as the arginine paradox. Possible reasons for the paradox include the restoration of impaired l-arginine transport, and the likelihood that arginine acts as an antioxidant. Although NO was not measured in the present study, we should not overlook the work of several investigators that show an increase in NO levels in arginine-supplemented animals and humans. l-Arginine has also been shown to prevent oxygen radical-induced dysfunction by either scavenging oxygen radicals (Lass et al. 2002) or delaying the formation of superoxide through electronic interaction with the haem-bound oxygen (Berka et al. 2004). Studies also indicate that dietary l-arginine increases the concentration of tetrahydrobiopterin (BH4) in the endothelial cells and improves vascular function (Kohli et al. 2004). BH4 is an essential cofactor in the production of NO (Schmidt et al. 1992; Meininger et al. 2000). In in vivo studies, Kohli et al. (2004) demonstrated that dietary arginine increases BH4 concentration in endothelial cells.

In conclusion, our results suggest that eNOS and iNOS play different roles in the development of chronic liver injury. We hypothesize that NO might possess both protective and detrimental functions in liver injury depending on the concentration and duration of NO production. Furthermore, NO derived from eNOS and iNOS plays a distinctive role in mediating liver injury with the altered eNOS expression being an important factor in mediating the progression of liver fibrosis. The results in this study agree with the observations that chronic liver injury induced by iNOS is mediated through a high level of NO production which causes oxidative stress and leads to cellular damage. In contrast, reduced NO production in the sinusoids due to reduced expression or activity of eNOS causes vasoconstriction and a probable decrease in blood supply to the liver. In addition, the present study also demonstrates that l-arginine was more effective than SMT in reducing CCl4-induced fibrosis. The beneficial effects of l-arginine on liver fibrosis might be due to its ability in maintaining a sustainable level of eNOS-derived NO production from the sinusoidal endothelium and thus keeping a normal hepatic blood flow as well as its antioxidant effect. Further investigation into the regulation of eNOS activity might provide more information on the effect of l-arginine on liver cirrhosis.

Taken together, the current study suggests that the progression of liver fibrosis is a result of a change in the expression of both iNOS and eNOS. Increased iNOS expression generates oxidative stress and a proinflammatory state while the reduced expression of eNOS affects the hepatic microcirculation. The effectiveness of the administration of l-arginine in attenuating the progression of liver fibrosis indicates that the impaired expression of eNOS in the sinusoids might be one factor in fibrotic liver injury, although the role of iNOS cannot be excluded.

Acknowledgments

This study was partly supported by grants from the Committee of Research and Conference Grants, The University of Hong Kong, Hong Kong, and the National Institutes of Health (AA 12893).

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