Potential conflict of interest: Nothing to report.
Increased intrahepatic resistance in cirrhotic livers is caused by endothelial dysfunction and impaired formation of two gaseous vasodilators, nitric oxide (NO) and hydrogen sulfide (H2S). Homocysteine, a sulfur-containing amino acid and H2S precursor, is formed from hepatic methionine metabolism. In the systemic circulation, hyperhomocystenemia impairs vasodilation and NO production from endothelial cells. Increased blood levels of homocysteine are common in patients with liver cirrhosis. In this study, we demonstrate that acute liver perfusion with homocysteine impairs NO formation and intrahepatic vascular relaxation induced by acetylcholine in methoxamine-precontracted normal livers (7.3% ± 3.0% versus 26% ± 2.7%; P < 0.0001). In rats with mild, diet-induced hyperhomocystenemia, the vasodilating activity of acetylcholine was markedly attenuated, and incremental increases in flow induced a greater percentage of increases in perfusion pressure than in control livers. Compared with normal rats, animals rendered cirrhotic by 12 weeks' administration of carbon tetrachloride exhibited a greater percentage of increments in perfusion pressure in response to shear stress (P < 0.05), and intrahepatic resistance to incremental increases in flow was further enhanced by homocysteine (P < 0.05). In normal hyperhomocysteinemic and cirrhotic rat livers, endothelial dysfunction caused by homocysteine was reversed by perfusion of the livers with sodium sulfide. Homocysteine reduced NO release from sinusoidal endothelial cells and also caused hepatic stellate cell contraction; this suggests a dual mechanism of action, with the latter effect being counteracted by H2S. Conclusion: Impaired vasodilation and hepatic stellate cell contraction caused by homocysteine contribute to the dynamic component of portal hypertension. (HEPATOLOGY 2008.)
Acritical factor for the development of portal hypertension in liver cirrhosis is the increment in vascular resistance to portal blood flow.1 Although liver fibrosis represents the main causative factor, a vasculogenic component contributed by insufficient production and/or availability of vasodilators, mainly nitric oxide (NO), within the hepatic microcirculation makes an important contribution.2, 3
Homocysteine is a sulfur-containing amino acid not found in the regular diet and primarily generated from the essential amino acid methionine in a variety of tissues including the liver.4 Methionine is the precursor of S-adenosylmethionine, a methyl donor in a number of methylation reactions involving RNA, DNA, proteins, and lipids.4 Homocysteine is formed upon demethylation of S-adenosylmethionine and subsequent hydrolysis of S-adenosylhomocysteine and lies at the junction of two intersecting pathways,5 the pathway that converts the sulfur atom of methionine to cysteine and glutathione (transsulfuration pathway) and the pathway (coupled to cobalamine, folate, and betaine metabolism) that reconverts homocysteine to methionine (remethylation pathway).
Increased plasma levels of homocysteine due to loss of function mutation or heterozygosity of cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) represent a well-defined risk factor for cardiovascular, thrombotic, neurodegenerative, and pregnancy-associated diseases.6–11 In these conditions, hyperhomocysteinemia promotes endothelial dysfunction and impairs endothelial-dependent vasodilation.6–11
Inhibition of CBS and CSE expression/function is a common finding in patients with chronic liver disorders,12–15 leading to hyperhomocysteinemia in two-thirds of patients with cirrhosis, regardless of the etiology of liver damage.16, 17 Despite its high prevalence, there is no understanding of whether hyperhomocysteinemia is a causative factor of sinusoidal endothelial cell (SEC) dysfunction and impaired endothelium-dependent vasodilation in portal hypertension.3, 18, 19 Because in the systemic circulation20 homocysteine promotes endothelial dysfunction by inactivating the generation of the endothelium-derived NO, we speculated that hyperhomocysteinemia could interfere with NO generation by SEC, the main source of NO in the hepatic circulation.18
Hydrogen sulfide (H2S) is a gaseous neuromodulator that exerts potent vasodilatory effects in both the systemic and splanchnic circulation.13, 14 In liver cells, H2S is generated from methionine and L-cysteine by the activity of CBS and CSE,5, 14 although alternative sources (for example, by activity of cysteine aminotransferase and/or 3-mercapto-sulfurtransferase) cannot be discounted.14 In addition to hepatocytes, hepatic stellate cells (HSCs), but not SEC, generate bioactive H2S from L-cysteine.13 Similarly to NO,1, 21 H2S counteracts HSC contraction induced by thrombin and attenuates the hepatic vasoconstriction caused by metoxamine in the normal and cirrhotic isolated and perfused liver.13 Because H2S represents the end product of methionine and L-cysteine metabolism, its liver content is reduced in rodent models of liver cirrhosis.13
In the present study, we have investigated whether homocysteine modulates the hepatic microcirculation in normal and cirrhotic livers and provided evidence that hyperhomocysteinemia impairs NO formation from SECs and causes HSC contraction.
All studies were approved by the Animal Study Committee of the University of Perugia. Male Wistar rats, 225-275 g, were from Charles River Breeding Laboratories (Monza, Italy) and were maintained on standard laboratory rat chow on a 12-hour light/dark cycle. Moderate hyperhomocysteinemia was induced in male Wistar rats by administration of L-methionine (1 g/kg of body weight/day) by gavage for a period of 3 weeks (n = 20).22 Liver cirrhosis was induced by bile duct ligation (BDL) and carbon tetrachloride (CCl4). BDL was performed as previously described,13 and all perfusion studies were performed 4 weeks after BDL. For the CCl4 model, rats were administered phenobarbital sodium (35 mg/dL) with drinking water for 3 days, followed by intraperitoneal injection of 100 μL of CCl4/100 g body weight in an equal volume of paraffin oil twice a week for 12 weeks.21
Evaluation of Hepatic Endothelial Dysfunction Induced by Homocysteine: Response to Acetylcholine and to Shear Stress
In normal, hyperhomocysteinemic and cirrhotic (BDL and CCl4) rats, the intrahepatic microcirculation was preconstricted with the α1-adrenergic agonist methoxamine (10−4 M), and 5 minutes later, concentration-effect curves13 to cumulative doses of acetylcholine (from 10−7 to 10−5 mol/L) were constructed.3, 13 The concentration of acetylcholine was increased by 1 log unit every 1.5 minutes. The response to cumulative doses of acetylcholine was calculated as a percentage change in perfusion pressure as previously described.3, 13 To test the effect of homocysteine, normal, hyperhomocysteinemic and cirrhotic livers were perfused with homocysteine (1 mmol/L) for 20 minutes before pressure/response curves were performed. In a separate set of experiments, the response to shear stress was tested. For this purpose, the perfusate was recirculated initially at 20 mL/minute and incrementally increased to 60 mL/minute over 25 minutes with continuous perfusion pressure monitoring.13 Homocysteine was added to the perfusate reservoir 25 minutes before the incrementally increased flow. To verify whether H2S reverses the homocysteine-induced effects, increasing concentrations of sodium sulfide (NaHS; from 0.1 to 1 mmol/L) were added to the perfusate 5 minutes before the pressure curves were performed.13 Global viability of the livers and liver weights were assessed as described previously at the end of perfusion.13
In Vitro Studies
Isolation and Culture of HSCs and SECs.
Rat hepatocytes and nonparenchymal cells were isolated as previously described.3, 13, 18 HSCs were cultured on plastic plates in Dulbecco's modified minimal essential medium containing 10% fetal bovine serum (FBS). Human SECs (Sciencecell Research Laboratories, San Diego, CA) were used for these studies as described previously.13
HSC contraction was induced by the addition of 20% FCS to the HSC monolayers.13 To assess whether homocysteine induced HSC contraction, HSCs were incubated with 50 and 100 μmol/L homocysteine for 18 hours, and contraction was measured. To test whether H2S inhibited homocysteine-induced contraction, NaHS (100 μmol/L) was added to the HSC monolayers. To allow contraction, lattices were detached by gentle circumferential dislodgment with a 200-μL micropipette tip, and contraction was measured by monitoring of the change in the lattice area over 18 hours.13
Western Blotting Analysis
Cultured SECs were scraped in HBSS, and the pellet was homogenized in a lysis buffer [50 mM trishydroxymethylaminomethane-HCl, 0.1 mM ethylene glycol tetraacetic acid, 0.1% (vol/vol) β-mercaptoethanol containing 100 mM leupeptin, 1 mM phenyl methyl sulfonyl fluoride, and 1% (vol/vol) Nonidet P40, pH 7.5]. After the removal of unbroken cells by centrifugation at 1.000g, the supernatant was boiled in Laemmli loading buffer and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis on a 7.5% acrylamide gel. Proteins were electroblotted onto nitrocellulose, and the membranes were washed in trishydroxymethylaminomethane-buffered saline with 0.1% Tween and blocked in 5% milk as described previously.18 Membranes were loaded with 100 μg of protein per lane and were subsequently incubated with a monoclonal antibody to endothelial nitric oxide synthase (eNOS) and a polyclonal antibody to caveolin-1 (Transduction Laboratories, Lexington, KY), and proteins were detected with a horseradish peroxidase–labeled anti-rabbit secondary antibody followed by enhanced chemiluminescence.
Determination of Liver and Plasma Concentrations of Homocysteine and H2S
Homocysteine was determined in blood samples collected from cardiac puncture of fasting rats with high-pressure liquid chromatography as previously described (Varian, Milan, Italy).23
H2S generation from liver tissues and plasma samples was measured according to a previously published method,13 Briefly, 100-150 mg liver samples were homogenized in 1 mL of ice-cold T-PER protein extraction reagent. Two milliliters of the mixture contained 10 mmol/L L-cysteine, 2 mmol/L pyridoxal 5′-phosphate, 100 mmol/L potassium phosphate buffer (pH = 7.4), and 10% (wt/vol) liver homogenates. Calmodulim and calcium chloride were added to the final reaction mixture at concentrations of 9.6 μmol/L and 0.6 mmol/L. Reactions were initiated by transfer of the tube from the ice bath to a 37°C water bath under a constant stream of nitrogen, which carried the sulfide acid in the second reactor containing 2 mL of a sulfide antioxidant buffer solution consisting of 2 mol/L KOH, 1 mol/L salicylic acid, and 0.22 mol/L ascorbic acid at pH 12.8. After incubation at 37°C for 90 minutes, 1 mL of a 50% trichloroacetic acid solution was added to the mixture to stop the reaction. The concentration of H2S in the sulfide antioxidant buffer solution was measured with a sulfide-sensitive electrode (model 9616 S2−/Ag+ electrode, Orion Research, Beverly, MA) as described.13
Measurement of NO Formation
Livers isolated from normal, methionine-fed and cirrhotic animals were perfused in situ in a recirculating system (150 cc of Krebs solution) at different flow rates as previously detailed.13 Samples of perfusate were obtained at 2 and 22 minutes before the pressure response curves were performed and subsequently every 15 minutes. Nitrates/nitrites (NOx) were measured as previously described.13, 21 For the in vitro experiment, levels of NOx were assessed in the supernatant of SEC cultures by chemiluminescence under the basal condition and after stimulation with bradykinin (10 μmol/L).18
Acetylcholine, methoxamine, DL-homocysteine, and all other chemicals were from Sigma (St. Louis, MO). Drugs were freshly prepared in distilled water.
All data are reported as means ± standard error of the mean. Statistical analysis was performed with the unpaired t test for the comparison of means of two groups. Concentration-effect curves were analyzed with analysis of variance (ANOVA) for repeated measures with the Bonferroni correction for multiple comparisons when appropriate. Significance was established at P < 0.05.
Homocysteine Causes Endothelial Dysfunction in Normal Livers
Perfusion of normal livers with acetylcholine produces a dose-dependent relaxation of methoxamine-precontracted hepatic microcirculation. At the highest dose, 10 μmol/L, acetylcholine caused a 26% ± 2.7% relaxation of methoxamine-induced contraction (Fig. 1A). Acute liver perfusion with homocysteine markedly attenuated the endothelium-dependent vasorelaxation induced by acetylcholine. When the two concentration-response curves were compared, it was evident that liver perfusion with homocysteine caused a robust attenuation of vasorelaxation induced by acetylcholine (P < 0.0001 by ANOVA).
To confirm the specific endothelial dysfunction induced by homocysteine, pressure-response curves were constructed in normal rat livers perfused with vehicle or homocysteine (Fig. 1B). In the homocysteine-perfused livers, intrahepatic resistance to incremental increases in flow was significantly enhanced in comparison with controls (n = 8 rats per group; P < 0.05). In addition, as shown in Fig. 1C, homocysteine-treated livers generated less NOx in response to incremental increases in flow in comparison with control livers (n = 8 rats per group; P < 0.05), and this indicates that acute perfusion with homocysteine impairs NO formation.
Diet-Induced Mild Hyperhomocysteinemia Is Associated with Liver Endothelial Dysfunction
Previous studies have shown that diet-induced mild hyperhomocysteinemia impairs systemic vasorelaxation.22 To investigate whether such an effect manifests also in the liver, mild hyperhomocysteinemia was induced by methionine being fed to rats22 Even though methionine feeding (3 weeks) increased plasma and liver homocysteine levels in comparison with normal diet-fed rats (Fig. 2B,2C; P < 0.05), H2S generation by liver tissues was unchanged (Fig. 2D). Methionine feeding also resulted in a significant increase in liver weight in comparison with control rats (10.1 ±0.1 versus 12.1 ± 0.6 g; n = 8; P < 0.05) and impaired intrahepatic vasorelaxation in response to cumulative doses of acetylcholine (n = 8 rats per group; P < 0.05 versus normal diet). Livers obtained from methionine-fed rats perfused with incremental increases in flow also exhibited a greater increment in the portal perfusion pressure and lower production of NOx in comparison with liver obtained from normal diet–fed rats (Fig. 2B,C; P < 0.05 versus normal diet). Thus, diet-induced mild hyperhomocysteinemia was associated with altered vasomotor function and endothelial dysfunction of the intrahepatic microcirculation.
Rodent Models of Liver Cirrhosis Are Associated with Severe Hyperhomocysteinemia and Reduced Generation of H2S
As illustrated in Fig. 3A,B, liver cirrhosis induced by CCl4 administration (and BDL; data not shown) was associated with increased plasma levels of homocysteine and reduced liver generation of H2S, the end product of homocysteine metabolism. Because these alterations were more pronounced in CCl4-treated rats, this model was used for the following experiments. CCl4 administration resulted in mild fibrosis and increased liver weight in comparison with control rats (10.1 ±0.1 versus 12.7 ± 0.1 g; n = 8; P < 0.001). As shown in Fig. 3C, CCl4 livers exhibited greater increases in portal perfusion pressure than control rats in response to incremental increases in flow (n = 6 rats per group; P < 0.05). In this experimental setting, perfusion of livers with homocysteine caused a further enhancement of intrahepatic resistance in response to incremental increases in flow, resulting in a steeper pressure increase in response to flow compared to vehicle-perfused cirrhotic livers (P < 0.001 by ANOVA). Compared with control livers, CCl4-treated rats exhibited a significant reduction of NOx release in response to incremental increases in flow (Fig. 3D; n = 4 rats per group; P < 0.001). This effect was slightly amplified, though in a nonsignificant way, by perfusion of the liver with homocysteine. Similar findings were observed in homocysteine-perfused BDL livers (data not shown).
H2S Corrects Endothelial Dysfunction Induced by Homocysteine in Normal, Homocysteinemic and Cirrhotic Rat Livers
We have examined whether H2S reverses endothelial dysfunction induced by homocysteine. As shown in Fig. 4A, perfusion of normal rat livers with NaHS protected against homocysteine-induced endothelial dysfunction (n = 8; P < 0.05 versus control), and the same vasodilating effect was evident also in the chronic nutritional mild hyperhomocysteinemia model (Fig. 4B; n = 8 rats; P < 0.05 versus control). In addition, liver perfusion with NaHS completely abrogated changes in intrahepatic resistance caused by homocysteine in CCl4-administered rats (Fig. 5A; n = 6-8; P < 0.05 versus CCl4 + homocysteine). Because the vasodilating effect of H2S was not associated with changes in NOx production (Fig. 5B; n = 6-8; P < 0.05 versus CCl4 + homocysteine), it appears that H2S can compensate for defective NO production induced by homocysteine in this animal model. Similar observations were made in BDL livers (data not shown).
Homocysteine Impairs NO Release by SECs and Induces HSC Contraction
Data shown in Fig. 6 provide support that, although exposure to homocysteine (50 μmol/L) did not cause any detectable change in SEC morphology, it resulted in a decline in the responsiveness of cells to eNOS activation induced by 10 μmol/L bradykinin (Fig. 6A; P < 0.05 versus untreated). However, homocysteine had no effect on eNOS or caveolin-1 protein levels, as measured by western blot analysis (Fig. 6B).
HSCs play an important role in regulating intrahepatic resistance to flow, relax in response to NO, and express the two major enzymes involved in homocysteine metabolism, CSE and CBS.1, 2, 13 As illustrated in Fig. 7 homocysteine caused a concentration-dependent contraction of HSCs, and this effect was reversed by coincubation of the cells with 100 mmol/L NaHS (n = 6; P < 0.05 versus homocysteine alone).
Homocysteine, an intermediary product of methionine metabolism,5 is a negative regulator of NO bioactivity in endothelial cells, and hyperhomocysteinemia is a well-recognized risk factor for cardiovascular disorders.6–11, 22 A dysregulated methionine metabolism occurs in two-thirds of patients with liver cirrhosis.12, 16, 17, 24 Inhibition of the expression/function of CBS and CSE, two genes critically involved in homocysteine breakdown to L-cysteine, is found in rodent models of liver cirrhosis,13 and loss of function mutation of CBS in mice results in altered hepatic function.25, 26
Here we have demonstrated that hyperhomocysteinemia induces endothelial dysfunction and impaired endothelium-dependent vasodilation in normal and cirrhotic rat livers. Support of this view comes from the demonstration that acute perfusion of normal rat liver with homocysteine impairs hepatic vasodilation induced by acetylcholine and shear stress and that this effect correlates with reduced NOx generation in the hepatic micrcirculation.1, 3 The enhanced intrahepatic resistance documented in normal livers exposed to homocysteine is consistent with the enhanced sensitivity to endothelial dysfunction of both the aorta25–30 and mesenteric arterioles obtained from mice with hyperhomocysteinemia induced by targeted disruption and heterozygosity of the CBS gene.27–30
Further supporting a role for homocysteine in the regulation of hepatic microcirculation, we found that mild chronic nutritional hyperhomocysteinemia21 reduces the hepatic vasodilation induced by acetylcholine and shear stress and impairs NO formation from SECs, the main source of NO in the hepatic microcirculation.18, 31
Under physiological conditions, the SECs have vital functions in regulating the vascular tone,1, 18 and the impairment of the endothelium-dependent vasodilatation is a sensitive indicator of endothelial dysfunction and a marker of increased intrahepatic resistance of liver cirrhosis. One of the key physiological functions of SECs is the generation of NO.1 In the liver microcirculation, NO acts as an endothelium-derived relaxing factor for vascular smooth muscle cells and HSCs.1, 3, 21, 31, 32 Homocysteine impairs NO release induced by bradykinin in SEC cultures, and this indicates that eNOS is a potential target for homocysteine in the liver microcirculation.1, 3, 18, 21, 31, 32 However, exposure of SEC to homocysteine had no effect on eNOS protein expression, as assessed by western blot analysis, nor did it increase the interaction of eNOS with caveolin-1, the major negative regulatory protein for eNOS activity in the liver microcirculation.33, 34 Thus, the mechanism through which homocysteine impairs NO bioavailability in SEC remains to be identified, although it is thought that homocysteine-induced oxidative stress might be involved.30
An important finding of the present study is the demonstration that homocysteine negatively regulates the ability of the hepatic microcirculation to respond to vasodilatory agents also in the cirrhotic livers. This finding was somewhat unexpected because increased intrahepatic resistance in these models is associated with a reduced ability of SEC to generate NO.1–3, 18, 31, 32 Thus, the observation that perfusion of CCl4livers with homocysteine causes a further deterioration of endothelial function but only partially decreases NO formation suggests that mechanisms other than NO inhibition are involved in the detrimental effect of homocysteine in these models. One possible explanation could be that the hyperhomocystenemia triggers a local release of vasoconstrictors.30 In support of this concept, it has been demonstrated that the combined metabolic burden of homocysteine and glucose stimulates endothelin-1 synthesis from bovine aortic endothelial cells.35
In addition to causing homocysteine accumulation, another functional consequence of reduced expression/function of CSE and CBS in the liver is a defective generation of H2S, the end product of homocysteine/L-cysteine metabolism.13 This gaseous mediator is an important vasodilatory agent for the hepatic microcirculation,13, 14 and perfusion of cirrhotic livers with H2S compensates for defective NO production in rodent models of portal hypertension.13 In the present study, we have confirmed that liver cirrhosis induced by CCl4 administration, but not methionine feeding, is associated with reduced generation of H2S and demonstrated that exogenous H2S reverses the vasoconstriction caused by homocysteine. This beneficial effect was NO-independent because liver perfusion with NaHS failed to increase NOx generation in normal and cirrhotic isolated and perfused livers. In the search for a mechanism or mechanisms that could have explained the protective effect of H2S in this model, we have demonstrated that, in addition to inhibition of NO formation by SEC, homocysteine triggers HSC contraction in vitro. This homocysteine-induced contraction of HSCs is counterbalanced by H2S. Because HSC contraction has relevance in regulating intrahepatic resistance,1, 2 these data suggest that HSCs might be a target for homocysteine and H2S.
In summary, we have demonstrated that homocysteine might contribute to the defective NO bioavailability in the liver microcirculation. Our data show that endothelial dysfunction contributed by homocysteine is compensated for by H2S in a NO-independent manner. These findings ground the concept that NO and H2S might exert additive effects in the liver microcirculation.