Effect of Lactobacillus-fermented diets on bacterial translocation and intestinal flora in experimental prehepatic portal hypertension.

Spontaneous bacterial infections in cirrhosis and portal hypertension have been attributed to translocation of gut-derived bacteria, a process promoted by intestinal bacterial overgrowth and disruption of the gut mucosal barrier. Bacteriotherapy with Lactobacillus has been reported to correct bacterial overgrowth, stabilize mucosal barrier function, and decrease bacterial translocation in rat models of acute liver injury and failure. In this study we investigated the effect of Lactobacillus-supplemented diets on intestinal flora and on bacterial translocation rate in portal vein ligated rats. Lactobacillus-fermented milk (yogurt) containing at least 2 x 10(9) colony forming units/ml or placebo (water) was adminstrated by gavage twice daily (2 ml) for 9 days. Portal vein ligation was performed on day 7 of treatment. Bacterial translocation to mesenteric lymph nodes and quantification of intestinal flora was assessed by standard bacteriological cultures. Bacterial translocation was not significantly different between animals that received yogurt (82%) and those that received placebo (75%). Yogurt did not induce any significant changes in intestinal flora, whether it was produced with Lactobacillus acidophilus or Lactobacillus GG. In conclusion, in acute prehepatic portal hypertension, bacteriotherapy with Lactobacillus was unable to induce changes in bacterial translocation probably because it was unable to induce changes in bacterial flora.

Bacterial translocation in cirrhotic rats stimulates eNOS-derived NO production and impairs mesenteric vascular contractility.

Nitric oxide (NO) has been implicated in the arterial vasodilation and associated vascular hyporesponsiveness to vasoconstrictors observed in liver cirrhosis. Bacteria, potent activators of NO and TNF-alpha synthesis, are found in the mesenteric lymph nodes (MLNs) of ascitic cirrhotic rats. Here, we investigated the impact of bacterial translocation (BT) to MLNs on TNF-alpha production, vascular NO release, and contractility in the mesenteric vasculature of ascitic cirrhotic rats. Vascular response to the alpha-adrenoagonist methoxamine, which is diminished in the superior mesenteric arterial beds of cirrhotic rats, is further blunted in the presence of BT. BT promoted vascular NO release in cirrhotic rats, an effect that depended on pressure-induced shear stress and was blocked by the NO inhibitor N(omega)-nitro-L-arginine. Removing the endothelium had the same effect. Endothelial NO synthase (eNOS), but not the inducible isoform (iNOS), was present in mesenteric vasculature of cirrhotic rats with and without BT, and its expression was enhanced compared with controls. TNF-alpha was induced in MLNs by BT and accumulated in parallel in the serum. This TNF-alpha production was associated with elevated levels of tetrahydrobiopterin (BH(4)), a TNF-alpha-stimulated cofactor and enhancer of eNOS-derived NO biosynthesis and NOS activity in mesenteric vasculature. These findings establish a link between BT to MLNs and increased TNF-alpha production and elevated BH(4) levels enhancing eNOS-derived NO overproduction, further impairing contractility in the cirrhotic mesenteric vasculature.

Impaired endothelial nitric oxide synthase activity associated with enhanced caveolin binding in experimental cirrhosis in the rat.

BACKGROUND & AIMS: A reduction in nitric oxide (NO) has been implicated as a cause of intrahepatic vasoconstriction in cirrhosis, but the regulatory mechanisms remain undefined. The aim of this study was to examine a contributory role for caveolin-1, a putative negative regulator of endothelial NO synthase, in mediating deficient intrahepatic NO production in the intact cirrhotic liver. METHODS: Cirrhosis was induced by carbon tetrachloride inhalation. Flow regulation of NO production and perfusion pressure was examined in the perfused rat liver. Protein expression of endothelial NO synthase (eNOS), caveolin, and calmodulin was examined by Western blotting and immunohistochemistry. NOS activity and NO production were assessed by citrulline generation and chemiluminescence, respectively. Protein-protein interactions were examined using whole tissue protein immunoprecipitation. RESULTS: In response to incremental increases in flow, cirrhotic animals produced significantly less NO(x) than control animals. NOS activity was significantly reduced in liver tissue from cirrhotic animals compared with control animals in the presence of similar eNOS protein levels. Deficient eNOS activity was associated with a severalfold increase in binding of eNOS with caveolin. Protein levels of caveolin-1 were markedly increased in the cirrhotic liver. CONCLUSIONS: These studies provide evidence that enhanced expression and interaction of caveolin with eNOS contribute to impaired NO production, reduced NOS activity, and vasoconstriction in the intact cirrhotic liver.

Hsp90 regulation of endothelial nitric oxide synthase contributes to vascular control in portal hypertension.

The molecular chaperone, heat shock protein 90 (Hsp90), acts as an intermediate in the signaling cascades leading to activation of endothelial nitric oxide synthase (eNOS). In this study, we examine the participation of this pathway in nitric oxide (NO)-dependent vasodilation in the rat mesentery in vitro. In normal animals, immunoprecipitation of eNOS from intact mesentery coimmunoprecipitates Hsp90 and, additionally, both eNOS and Hsp90 colocalize to the endothelial lining of mesenteric vessels. In the perfused mesenteric vasculature of normal animals, geldanamycin (GA), a specific inhibitor of Hsp90 signaling, attenuates ACh-dependent vasodilation but does not affect vasodilation in response to sodium nitroprusside. Next, studies were performed in animals with experimental portal hypertension induced by portal vein ligation (PVL). In PVL animals, NOS catalytic activity is markedly enhanced in mesenteric tissue and the perfused mesentery is hyporesponsive to the vasoconstrictor methoxamine (MTX). GA significantly potentiates MTX-induced vasoconstriction after PVL, thereby partially reversing the hyporeactivity to this agent exhibited in the mesenteric vasculature after PVL. These studies suggest that Hsp90 can act as a signaling mediator of NO-dependent responses in the mesenteric circulation and indicate that the excessive NO production observed in portal hypertension is mediated in part through Hsp90 signaling.

Interleukin-6, nitric oxide, and the clinical and hemodynamic alterations of patients with liver cirrhosis.

OBJECTIVE: Nitric oxide has been proposed as a mediator of hyperdynamic circulation in cirrhosis. Endotoxin and cytokines induce the synthesis of nitric oxide. The aim of this study was to investigate the relationship between endotoxemia, cytokines, and nitric oxide in patients with cirrhosis, and to correlate these findings with clinical, biochemical, and hemodynamic parameters. METHODS: Clinical, biochemical, and hemodynamic parameters were assessed in 66 patients with cirrhosis and 15 controls. Levels of antidiuretic hormone, plasma renin activity, aldosterone, interferon gamma, interleukin-1, interleukin-6, tumor necrosis factor alpha, endotoxin, and nitrates-nitrites were determined. RESULTS: Mean arterial pressure was lower and interleukin-6, tumor necrosis factor alpha, nitrites-nitrates levels, and endotoxin positivity rates were higher in cirrhotics than in controls (p < 0.005). Mean arterial pressure decreased and interleukin-6 levels increased with worsening of Child score (p < 0.005). Patients with ascites had higher levels of interleukin-6, tumor necrosis factor alpha, and nitrates-nitrites than patients without ascites (p < 0.01). Elevated levels of interleukin-6 were found in patients with encephalopathy grade I, compared with patients without (p < 0.001); this association was independent of the severity of liver disease. In patients with low mean arterial pressure, interleukin-6 levels were higher than in patients with high mean arterial pressure (p = 0.001), whereas tumor necrosis factor alpha and nitrates-nitrites levels were not different. By multivariate analysis, high interleukin-6 levels showed independent associations with the presence of ascites, encephalopathy, and low mean arterial pressure. Only interleukin-6 levels had significant correlations with Child score, plasma renin activity, serum and urinary sodium, and mean arterial pressure (r > or = 0.4, p < 0.005). CONCLUSIONS: Although the activity of the nitric oxide pathway is increased in patients with cirrhosis and might contribute to the hemodynamic alteration, other factors are involved. Interleukin-6, possibly through nitric oxide-independent mechanisms, also might play a role in the vasodilatation of cirrhosis and the pathogenesis of hepatic encephalopathy.

The diagnostic and predictive value of ascites nitric oxide levels in patients with spontaneous bacterial peritonitis.

Nitric oxide (NO) is a messenger molecule involved in pathogen suppression. Cirrhosis is characterized by an increased risk for infections, including spontaneous bacterial peritonitis (SBP). The role of NO in the infections that develop in cirrhosis has not been clearly established. The aim of this study was to investigate the utility of measuring ascites NO in the diagnosis of SBP and/or in determining the predisposition of cirrhotic patients to develop this infection. Nitric oxide metabolites (nitrites + nitrates [NOx]) were measured by chemiluminescence in 105 ascites samples obtained from 87 cirrhotic patients and in 87 simultaneously obtained serum samples. Ascites NO levels were not significantly different among ascites from patients with SBP (n = 39; median, 48 micromol/L), patients with sterile ascites (n = 54; median, 42 micromol/L), and samples obtained after patients with SBP had been treated (n = 12; median, 62 micromol/L). No differences in ascites NO levels were observed between culture-positive and culture-negative peritonitis. Among 50 patients with sterile ascites on initial paracentesis, 7 patients developed peritonitis during follow-up; no differences in baseline NO levels were observed between patients who developed peritonitis (median, 46 micromol/L) and those who did not (median, 41 micromol/L). Among patients with SBP, mortality was significantly higher in those with NO levels >60 micromol/L. A very significant direct correlation was found between ascites and serum NO levels (r2 = .86). In conclusion, ascites NO levels in cirrhotic patients are not useful either to diagnose or to determine predisposition to SBP. Rather, ascites NO levels reflect serum levels, are higher in cirrhotic patients with more severe liver disease, and may be a useful prognostic marker.

Thalidomide inhibits tumor necrosis factor alpha, decreases nitric oxide synthesis, and ameliorates the hyperdynamic circulatory syndrome in portal-hypertensive rats.

A hyperdynamic circulatory state frequently is observed in portal hypertension with liver failure or extensive portal-systemic shunting. Tumor necrosis factor alpha (TNF) causes marked hypotension in mammals by inducing nitric oxide synthesis and has been shown to play a role in the development of the hemodynamic changes observed in portal hypertension. Thalidomide selectively inhibits TNF production by enhancing messenger RNA degradation. We investigated the systemic and portal hemodynamic effects of thalidomide in a prehepatic model of portal hypertension and evaluated whether suppressing TNF synthesis decreases NO production. Portal hypertension was induced by partial ligation of the portal vein (PVL). Animals received thalidomide (T) (50 mg/kg/d) + water or water alone (W), orally, daily for 2 days before and 13 days after PVL operation, at which time hemodynamic studies were performed and TNF plasma levels were obtained. Sham-operated animals were studied identically. In an additional group of PVL animals, 24-hour urinary excretion of NO2- and NO3- was measured during treatment. PVL animals receiving T presented with a significantly higher mean arterial pressure and systemic vascular resistance and significantly lower portal pressure, TNF plasma levels, and 24-hour urinary excretion of NO2- and NO3-, in comparison with rats receiving W. A significant correlation (r = -0.61) was observed between TNF plasma levels and mean arterial pressure among PVL animals. Thalidomide did not have any significant effects on sham rats. Thalidomide inhibits TNF synthesis and reduces NO production, blunts the development of the hyperdynamic circulation, and decreases portal pressure in PVL-operated rats.