Soluble guanylyl cyclase stimulation and phosphodiesterase-5 inhibition improve portal hypertension and reduce liver fibrosis in bile duct-ligated rats.

BACKGROUND: In cirrhosis, the nitric oxide-soluble guanylyl cyclase (sGC)-cyclic guanosine monophosphate (cGMP) pathway is impaired, which contributes to increased intrahepatic vascular resistance (IHVR) and fibrogenesis. We investigated if sGC stimulation (riociguat (RIO)), sGC activation (cinaciguat (CINA)) or phosphodiesterase (PDE)-5 inhibition (tadalafil (TADA)) improves portal hypertension (PHT) and liver fibrosis. METHODS: Fifty male Sprague-Dawley rats underwent bile-duct ligation (BDL) or sham operation. RIO (0.5 mg/kg), CINA (1 mg/kg), TADA (1.5 mg/kg) or vehicle (VEH) was administered from weeks 2 to 4 after BDL. At week 4, invasive haemodynamic measurements were performed, and liver fibrosis was assessed by histology (chromotrope-aniline blue (CAB), Picro-Sirius red (PSR)) and hepatic hydroxyproline content. RESULTS: Cirrhotic bile duct-ligated rats presented with PHT (13.1 ± 1.0 mmHg) and increased IHVR (4.9 ± 0.5 mmHg⋅min/mL). Both RIO (10.0 ± 0.7 mmHg, p = 0.021) and TADA (10.3 ± 0.9 mmHg, p = 0.050) decreased portal pressure by reducing IHVR (RIO: -41%, p = 0.005; TADA: -21%, p = 0.199) while not impacting heart rate, mean arterial pressure and portosystemic shunting. Hepatic cGMP levels increased upon RIO (+239%, p = 0.006) and TADA (+32%, p = 0.073) therapy. In contrast, CINA dosed at 1 mg/kg caused weight loss, arterial hypotension and hyperlactataemia in bile duct-ligated rats. Liver fibrosis area was significantly decreased by RIO (CAB: -48%, p = 0.011; PSR: -27%, p = 0.121) and TADA (CAB: -21%, p = 0.342; PSR: -52%, p = 0.013) compared to VEH-treated bile duct-ligated rats. Hepatic hydroxyproline content was reduced by RIO (from 503 ± 20 to 350 ± 30 µg/g, p = 0.003) and TADA (282 ± 50 µg/g, p = 0.003), in line with a reduction of the hepatic stellate cell activation markers smooth-muscle actin and phosphorylated moesin. Liver transaminases decreased under RIO (AST: -36%; ALT: -32%) and TADA (AST: -24%; ALT: -27%) treatment. Hepatic interleukin 6 gene expression was reduced in the RIO group (-56%, p = 0.053). CONCLUSION: In a rodent model of biliary cirrhosis, the sGC stimulator RIO and the PDE-5 inhibitor TADA improved PHT. The decrease of sinusoidal vascular resistance was paralleled by a reduction in liver fibrosis and hepatic inflammation, while systemic haemodynamics were not affected.

Folic acid ameliorates homocysteine-induced angiogenesis and portosystemic collaterals in cirrhotic rats.

INTRODUCTION AND OBJECTIVES: Liver cirrhosis is characterized by increased intrahepatic resistance, splanchnic vasodilation/angiogenesis, and formation of portosystemic collateral vessels. Collaterals can cause lethal complications such as gastroesophageal variceal hemorrhage. Homocysteine is linked to vascular dysfunction and angiogenesis and higher levels have been reported in cirrhotic patients. It is also known that folic acid supplementation reverses the effects of homocysteine. However, the treatment effect in cirrhosis has yet to be investigated. MATERIAL AND METHODS: Liver cirrhosis was induced in Sprague-Dawley rats with common bile duct ligation (CBDL). The CBDL rats randomly received (1) vehicle; (2) dl-homocysteine thiolactone (1g/kg/day); (3) dl-homocysteine thiolactone plus folic acid (100mg/kg/day); or (4) folic acid. On the 29th day, hemodynamic parameters, liver and renal biochemistry, protein expressions of proangiogenic factors, mesenteric vascular density and portosystemic shunting were evaluated. RESULTS: In the cirrhotic rats, homocysteine increased mesenteric vascular density and the severity of shunting. It also up-regulated the protein expressions of mesenteric vascular endothelial growth factor (VEGF) and phosphorylated-endothelial nitric oxide synthase (p-eNOS). These effects were reversed by folic acid treatment (P<0.05). CONCLUSION: Folic acid ameliorated the adverse effects of homocysteine in the cirrhotic rats, which may be related to down-regulation of the VEGF-NO signaling pathway.

Combined Enteral and Parenteral Glutamine Supplementation in Endotoxemic Swine: Effects on Portal and Systemic Circulation Levels.

OBJECTIVE: To measure plasma glutamine (GLN) levels in systemic and portal circulation after combined enteral and parenteral administration in early endotoxemic swine. We hypothesized that this combination will be more efficient than intravenous administration alone in restoring plasma levels during the course of endotoxemia. MATERIALS AND METHODS: Endotoxemia was induced with Escherichia coli O111:B4 lipopolysaccharide (LPS) (250 µg/kg body weight) in 16 anes-thetized, fasted swine and maintained by constant infusion (2 µg/kg/h) over 180 min. Another 16 swine served as controls. After infusion with LPS or placebo, GLN was administered intravenously, enterally or in combination (0.5 g/kg i.v. plus 0.5 g/kg enterally) over 30 min. At 0, 15, 30, 45, 60, 120 and 180 min, blood was drawn from the systemic and portal circulation for colorimetric assessment of GLN. RESULTS: In healthy, placebo-alone swine, GLN levels remained stable throughout the study. Intravenous and combined infusion increased systemic levels (p = 0.001), but after enteral administration alone, a smaller effect was observed (p = 0.026). Portal levels were increased after combined, enteral and intravenous administration (p = 0.001). In endotoxemia, systemic and portal levels decreased significantly. Intravenous and, to a greater extent, combined administration increased systemic levels (p = 0.001), while enteral administration only had a small effect (p = 0.001). In the portal vein, intravenous and combined treatment increased plasma levels (p = 0.001), whereas enteral supplementation alone had again a small, yet significant effect (p = 0.001). CONCLUSIONS: The findings indicate that combined GLN supplementation is superior to intravenous treatment alone, in terms of enhanced availability in systemic and portal circulations. Thus, combined treatment at the onset of endotoxemia is a beneficial practice, ensuring adequate GLN to compensate for the resulting intracellular shortage.

PPARγ ligand attenuates portal inflammation in the MRL-lpr mouse: a new strategy to restrain cholangiopathy in primary biliary cirrhosis.

Primary biliary cirrhosis (PBC) is characterized by chronic destructive cholangitis, which is associated with the reduced expression of an anti-inflammatory molecule, peroxisome proliferator-activated receptor-γ (PPARγ), in intrahepatic bile ducts. We previously demonstrated the anti-inflammatory effects of PPARγ ligands using cultured human biliary epithelial cells. In this study, we evaluated the effectiveness of PPARγ ligand against peribiliary inflammation in vivo. As an animal model of PBC, we used MRL/lpr mice in which a PBC-like cholangitis occurs naturally. Anti-inflammatory effects of the intraperitoneal administration of a PPARγ ligand, the prostaglandin D metabolite 15-deoxy-Δ(12,14)-prostaglandin J2 (15d-PGJ2), were evaluated. In untreated mice, portal inflammation including cholangitis was found to some degree in the majority of portal tracts. In mice given a high-dose group, the degree of portal inflammation was significantly reduced and mice mostly lacking portal inflammation and cholangitis were also found. T cell numbers in portal tracts were markedly decreased in the high-dose group, compared with controls, whereas there was no significant difference in terms of B cells and macrophages. This study is the first to assess the therapeutic potential of a PPARγ ligand against portal inflammation including cholangitis. Anti-inflammatory effects of PPARγ ligands may prevent the progression of cholangiopathy in PBC patients.

Levosimendan increases portal blood flow and attenuates intestinal intramucosal acidosis in experimental septic shock.

It has been proposed that vasodilatory therapy may increase microcirculatory blood flow and improve tissue oxygenation in septic shock. The authors aimed to evaluate the effects of levosimendan in systemic and splanchnic hemodynamics in a porcine model of septic shock in a randomized animal controlled study. This study was performed in an animal research facility in a university hospital. Anesthetized pigs were monitored with a pulmonary artery catheter and an ultrasonic blood flow probe in the portal vein for measurement of systemic and portal blood flows and with a tonometer placed in the small intestine for measurement of the intramucosal-arterial PCO2 gap. Three groups of pigs were studied: nonseptic (n = 7), septic (n = 7), and septic treated with levosimendan (n = 7). Levosimendan was administered i.v. at t = -10 min (200 microg/kg in i.v. bolus followed by 200 microg/kg per h). Sepsis was induced at t = 0 min by the administration of live Escherichia coli. Vascular reactivity was tested by the hemodynamic response to noradrenaline. Levosimendan markedly attenuated the sepsis-induced increase in pulmonary vascular resistance, decrease in portal/systemic blood flow, oliguria, impairment in oxygenation, hyperkalemia, and the widened intramucosal-arterial PCO2 gap. Systemic blood pressure and vascular resistance did not differ as compared with the septic untreated group. Responses to noradrenaline significantly improved in animals treated with levosimendan. Treatment with levosimendan in this animal model of sepsis attenuated pulmonary vasoconstriction and improved portal blood flow, intestinal mucosal oxygenation, pulmonary function, and vascular reactivity.

Effects of 2-hydroxy-4-methylthiobutyrate on portal plasma flow and net portal appearance of amino acids in piglets.

To determine whether portal plasma flow (PPF) and net portal appearance of amino acids (AA) could be affected by 2-hydroxy-4-methylthiobutyrate (HMB), six barrows (35-day-old, 8.6+/-1.4 kg), implanted with arterial, portal and mesenteric catheters, were fed a DL-methionine (as the control) or HMB-supplemented diet once hourly and infused intramesenterically with 1% p-amino hippurate. PPF was numerically 9% higher (P=0.09) in HMB-fed pigs than in controls over a 4-6 h period. Compared with controls, pigs fed the HMB diet had increased (P<0.05) net portal balance and/or appearance of leucine, isoleucine, histidine, arginine and alanine, but had decreased (P<0.05) portal appearance of glutamate over a 6-h period. The concentration of acetate in the lumen of the distal small intestine was higher (P=0.01) in HMB-fed pigs than in controls (25.14 vs. 7.64 mmol/kg). mRNA levels for proglucagon and endothelial nitric-oxide synthase (eNOS) in stomach and proximal small intestine, and mRNA levels for GLP-2 receptor (GLP-2R) in stomach were higher (P<0.05) in HMB-fed pigs compared with those in controls. Collectively, HMB supplementation increased concentrations of short-chain fatty acids in intestinal lumen, expression of proglucagon, GLP-2R, and eNOS genes, and net portal absorption of AA. These novel findings from the study with pigs may also have important implications for intestinal nutrition and health in humans.

Role of PGI2 in the formation and maintenance of hyperdynamic circulatory state of portal hypertensive rats.

AIM: To investigate the role of prostacyclin (PGI(2)) and nitric oxide (NO) in the development and maintenance of hyperdynamic circulatory state of chronic portal hypertensive rats. METHODS: Ninety male Sprague-Dawley rats were divided into three groups: intrahepatic portal hypertension (IHPH) group by injection of CCl(4), prehepatic portal hypertension (PHPH) group by partial stenosis of the portal vein and sham-operation control (SO) group. One week after the models were made, animals in each group were subdivided into 4 groups: saline controlled group (n = 23), Nomega-nitro-L-arginine (L-NNA) group (n = 21) group, indomethacin (INDO) group (n = 22) and high-dose heparin group (n = 24). The rats were administrated 1 mL of saline, L-NNA (3.3 mg/kg.d) and INDO (5 mg/kg.d) respectively through gastric tubes for one week, then heparin (200 IU/Kg/min) was given to rats by intravenous injection for an hour. Splanchnic and systemic hemodynamics were measured using radioactive microsphere techniques. The serum nitrate/nitrite (NO(2)(-)/NO(3)(-)) levels as a marker of production of NO were assessed by a colorimetric method, and concentration of 6-keto-PGF1alpha, a stable hydrolytic product of PGI(2), was determined by radioimmunoassay. RESULTS: The concentrations of plasma 6-keto-PGF1alpha (pg/mL) and serum NO(2)(-)/NO(3)(-) (micromol/L) in IHPH rats (1123.85+/-153.64, 73.34+/-4.31) and PHPH rats (891.88+/-83.11, 75.21+/-6.89) were significantly higher than those in SO rats (725.53+/-105.54, 58.79+/-8.47) (P<0.05). Compared with SO rats, total peripheral vascular resistance (TPR) and spanchnic vascular resistance (SVR) decreased but cardiac index (CI) and portal venous inflow (PVI) increased obviously in IHPH and PHPH rats (P<0.05). L-NNA and indomethacin could decrease the concentrations of plasma 6-keto-PGF1alpha and serum NO(2)(-)/NO(3)(-) in IHPH and PHPH rats (P<0.05). Meanwhile, CI, FPP and PVI lowered but MAP, TPR and SVR increased (P<0.05). After deduction of the action of NO, there was no significant correlation between plasma PGI(2) level and hemodynamic parameters such as CI, TPR, PVI and SVR. However, after deduction of the action of PGI(2), NO still correlated highly with the hemodynamic parameters, indicating that there was a close correlation between NO and the hemodynamic parameters. After administration of high-dose heparin, plasma 6-keto-PGF(1alpha) concentrations in IHPH, PHPH and SO rats were significantly higher than those in rats administrated vehicle (P<0.05). On the contrary, levels of serum NO(2)(-)/NO(3)(-) in IHPH, PHPH and SO rats were significantly lower than those in rats administrated Vehicle (P<0.05). Compared with those rats administrated vehicle, the hemodynamic parameters of portal hypertensive rats, such as CI and PVI, declined significantly after administration of high-dose heparin (P<0.05), while TPR and SVR increased significantly (P<0.05). CONCLUSION: It is NO rather than PGI(2) that is a mediator in the formation and maintenance of hyperdynamic circulatory state of chronic portal hypertensive rats.

Effect of neonatal capsaicin treatment on haemodynamics and renal function in cirrhotic rats.

BACKGROUND: Mechanisms underlying abnormalities of circulation and renal function in cirrhosis are not completely understood. Our previous study revealed that primary afferent denervation by neonatal capsaicin treatment prevented the development of hyperdynamic circulation in portal hypertensive and cirrhotic rats. AIMS: The present study aimed to clarify the role of capsaicin sensitive nerves in the development of renal dysfunction and ascites formation in cirrhosis. METHODS: Rat pups were injected with capsaicin (50 mg/kg) or vehicle and allowed to grow. When they reached adulthood, cirrhosis was induced by bile duct ligation while controls received sham operation. Cardiac output and regional blood flows were measured by radioactive microspheres, glomerular filtration rate by (3)H inulin clearance, and urine volume, sodium excretion, and ascites formation were determined. Immunohistochemical staining for Fos in the brain stem cardiovascular regulatory nuclei, the nucleus of the solitary tract, and ventrolateral medulla was measured as an index of central neuronal activation. RESULTS: Increased cardiac output and renal blood flow, and decreased systemic vascular resistance, arterial pressure, renal vascular resistance, and glomerular filtration rate, as well as ascites, were found in vehicle treated cirrhotic rats. Neonatal capsaicin treatment completely blocked the development of hyperdynamic circulation and ascites, and improved renal function in cirrhotic rats. This was associated with complete abrogation of brain stem neuronal activation in capsaicin treated cirrhotic rats. CONCLUSIONS: These results indicate that intact primary afferent innervation is necessary for the development of not only the hyperdynamic circulation but also the renal dysfunction and ascites formation characteristic of cirrhosis.

Mesenteric and hepatic vascular reactivity in Donryu rats with and without a cholesterol-supplemented diet.

Vascular function of Donryu rats fed on a normal or cholesterol-supplemented diet was examined in the isolated perfused mesenteric arterial bed and portally perfused liver. In mesenteric preparations, frequency-dependent vasoconstriction to electrical field stimulation (4-32 Hz, 1 ms, 90 V, 30 s) and dose-dependent vasoconstriction to noradrenaline (0.15-1500 nmol) was similar in both groups. Dose-dependent vasoconstriction to alpha, beta-methylene ATP (0.05-500 nmol) via P2x purinoceptors was significantly impaired in Donryu rats fed on a cholesterol-supplemented diet. In preparations with raised tone (methoxamine 5-35 microM), there was no significant difference in endothelium-dependent relaxation to acetylcholine and ATP, or endothelium-independent relaxation to sodium nitroprusside. In liver preparations, there was no difference in frequency-dependent vasoconstriction to electrical field stimulation (2-32 Hz, 1 ms, 90 V, 30 s), or dose-dependent vasoconstriction to noradrenaline (0.05-500 nmol) and alpha, beta-methylene ATP (0.05-500 nmol) between the groups. In conclusion, in mesenteric arteries, but not in the hepatic portal vasculature of Donryu rats fed on cholesterol P2x purinoceptor function is impaired, but sympathetic neurotransmission is unaffected. Mesenteric endothelial and smooth muscle function is unimpaired.

Effects of high-copper feeding on portal ammonia absorption and on oxygen consumption by portal vein-drained organs and by the whole animal in growing pigs.

Growing gilts that had catheters inserted into the portal vein, ileal vein, and carotid artery and that were trained to consume 1.2 kg of a 16% CP corn-soybean meal basal diet once daily were used. In Trial 1, hourly simultaneous measurements of the O2 consumption by portal vein-drained organs (PVDO) and by the whole animal during the 24- to 30-h postprandial period were conducted in eight pigs (33.8 +/- .6 kg). After initial measurements, four pigs continued to receive the basal diet and four pigs were fed a basal diet +250 ppm of Cu. Following a 7-d acclimation period, the second series of measurements were made. In pigs fed the diet supplemented with Cu, the PVDO and whole-animal O2 consumptions and the fraction of whole-animal O2 consumption used by PVDO were not different (P > .05) between the initial and second series. In Trial 2, seven gilts (38.5 +/- .9 kg) were used for measurements of net portal NH3 absorption and the O2 consumption by PVDO and by the whole animal during the 0- to 6-h postprandial period. The second series of measurements were made 7 d after four pigs were fed the diet supplemented with Cu. For pigs fed the diet supplemented with Cu, during the second series of measurements, the net portal NH3 absorption was lowered (P < .05). No differences (P > .05) in PVDO and whole-animal O2 consumptions and the fraction of whole-animal O2 consumption used by PVDO were found between the initial and second series.(ABSTRACT TRUNCATED AT 250 WORDS)

Dose-dependent effects of glycine, alanine, and glucose on hepatic bile secretion, oxygen consumption, and hemodynamics.

To assess the effects of a small (0.5%) and a large dose (5%) of glycine and alanine and of hypertonic glucose on hepatic bile secretion, oxygen consumption, and hemodynamics, experiments were performed on anesthetized pigs. Only the large dose of amino acids exerted significant changes. Glycine, alanine, and glucose reduced bile acid-dependent bile secretion gradually, which was nearly halved from a control value of 0.32 +/- 0.04 ml/min. Oxygen consumption was thereby continuously stimulated during amino acid and glucose infusion and increased from 448 +/- 132 mumol/min before to 995 +/- 226 mumol/min after the infusion of glycine, alanine, and glucose. Hepatic arterial blood flow increased from 214 +/- 14 ml/min to 238 +/- 14 ml/min after glycine infusion, whereas portal venous blood flow decreased from 542 +/- 50 ml/min to 481 +/- 47 ml/min. Total hepatic blood flow remained unchanged. Alanine and glucose provoked no further changes in hepatic blood flow. Bile secretion is a sensitive marker of hepatic metabolism, whereas hepatic blood flow is not a dominant regulator of bile secretion. Stimulation of hepatic metabolism is not followed by changes in total hepatic blood flow.

[The dose-dependent action of naloxone on systemic and portal circulation in acute blood loss in rats]. / Dozozavisimoe deistvie naloksona na sistemnoe i portal'noe krovoobrashchenie pri ostroi krovopotere u krys.

Using the method of contact luminescent biomicroscopy of the liver and the intestine coupled with the ultrasonic measurement of systemic blood pressure, blood flow velocity in the portal vein and hepatic artery it has been established that in rats with acute decompensatory hemorrhage naloxone increases blood pressure and improves the state of protal macro and microcirculation only after i. v. injection of large dose (5 mg/kg). Naloxone does not influence the dynamics of acute compensatory hemorrhage and the development of the posthemorrhagic microcirculatory disturbances (local microstases, microthromboses, erythrocyte aggregation).