Randomised clinical trial: the safety and efficacy of long-acting octreotide in patients with portal hypertension.

BACKGROUND: It remains unclear whether a long-acting preparation of octreotide (Sandostatin LAR) can be safely used for portal hypertension in patients with compensated cirrhosis. AIM: To determine the safety and efficacy of LAR among patients with Child Pugh Class A or B cirrhosis and small oesophageal varices. METHODS: A randomised, double-blind, placebo-controlled study was conducted in 39 patients with cirrhosis and small oesophageal varices. Safety was based on frequency and severity of adverse events. Efficacy was determined by hepatic vein pressure gradient (HVPG) measured at baseline and day 84 following administration of LAR 10 mg (n = 15), 30 mg (n = 10) or saline (n = 14). Fasting and postprandial portal blood flow (PBF), superior mesenteric artery pulsatility index (SMA-PI), glucagon and octreotide levels were measured. An intention-to-treat analysis was performed. RESULTS: Four patients in the LAR 30 group (40%) withdrew from the study due to serious adverse events. No patient in the LAR 10 or control group had serious adverse events. There was no statistically significant decrease between HVPG at day 84 and baseline with LAR 30 mg (11.8 ± 2.3 mmHg vs. 14.1 ± 3.2), LAR 10 mg (15.3 ± 4.8 mmHg vs. 15.1 ± 3.8), or saline (13.3 ± 3.8 mmHg vs. 15.1 ± 4.3) (P = 0.26). Neither PBF, SMA-PI nor plasma glucagon levels were significantly decreased from baseline (P = 0.56). CONCLUSIONS: The absence of significant haemodynamic benefit, as well as the high frequency of severe adverse events associated with use of LAR, do not support the use of this agent in the treatment of portal hypertension.

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.

Propranolol for the prevention of first esophageal variceal hemorrhage: a lifetime commitment?

Although beta blockers have had significant impact in the treatment of portal hypertension, the question of how long they should be continued for prevention of variceal hemorrhage remains unknown. Prospective studies on beta blockers to prevent variceal hemorrhage lack long-term follow-up, and indefinite administration of beta blockers for primary prevention of variceal bleeding has become standard practice. The aim of this study was to determine the outcomes of patients in whom beta blocker therapy was discontinued. Patients completing a prospective, randomized, double-blind, placebo-controlled trial of propranolol for the primary prevention of variceal hemorrhage were tapered off of propranolol and placebo and followed prospectively for subsequent events. Of the 49 patients in the follow-up study (25 former propranolol, 24 former placebo), 9 experienced variceal hemorrhage (6 former propranolol, 3 former placebo). Following withdrawal of propranolol, the freedom from variceal bleeding was not significantly different between these 2 groups of patients, suggesting that the protective effect of propranolol against variceal hemorrhage, noted previously, was no longer present. Seventeen patients died (12 former propranolol, 5 former placebo) during the follow-up study. Cumulative survival was longer in the placebo group. These trends for EVH and survival were opposite to those observed in the original study population while patients were taking medication. When propranolol is withdrawn, the risk of variceal hemorrhage returns to what would be expected in an untreated population. Patients who discontinue beta blockers experience increased mortality compared with an untreated population. These observations support the current practice of indefinite prophylactic therapy.

Octreotide potentiates PKC-dependent vasoconstrictors in portal-hypertensive and control rats.

BACKGROUND & AIMS: The effect of octreotide on vascular tone in the superior mesenteric artery (SMA) was studied in portal-hypertensive (portal vein-ligated) and sham-operated rats. METHODS: In vitro-perfused SMA vascular beds were tested for the cumulative dose-response to octreotide at baseline conditions and after preconstriction with different vasoconstrictors (alpha1-agonist methoxamine, endothelin [ET-1], phorbol ester [PdBu], and potassium chloride [KCl]). RESULTS: Octreotide did not affect baseline perfusion pressures (without preconstriction). alpha1-Adrenergic-, ET-1-, and PdBu-, but not KCl-, induced vasoconstriction was significantly potentiated by octreotide. This effect was dose-dependent and not different in portal vein-ligated and sham rats. Amplification of alpha1-adrenergic vasoconstriction by octreotide was significantly enhanced by nitric oxide inhibition (N(W)-nitro-L-arginine, 10(-4) mol/L) as well as by removal of the endothelium, and was completely suppressed by inhibition of protein kinase C (calphostin C, 1 micromol/L), phospholipase A2 (quinacrine, 5 micromol/L), and cyclooxygenase (indomethacin, 20 micromol/L). CONCLUSIONS: Not directly, but in the presence of vasoconstrictors involving activation of protein kinase C, octreotide exerts a local vasoconstrictive effect on vascular smooth muscle of SMA. This potentiation is equipotent in portal vein-ligated and sham rats, immediate in onset, and mediated via phospholipase A2 and cyclooxygenase-derived prostanoids. This indicates that in preprandial conditions octreotide enhances the vasoconstrictive effect of dependent vasoconstrictors.

Local arterial vasoconstriction induced by octreotide in patients with cirrhosis.

Peripheral vasodilation initiates the hyperdynamic circulation in cirrhosis. Somatostatin and its analogues, such as octreotide, have a vasoconstrictive effect in cirrhotic patients and experimental animals with portal hypertension. The exact mechanism of octreotide-induced vasoconstriction remains unknown. To investigate whether octreotide produces vasoconstriction through suppression of vasodilatory peptides, such as glucagon, or through a local effect, we evaluated the effect of an intra-arterial dose on forearm blood flow (FBF), while measuring systemic glucagon levels. FBF was measured in 10 cirrhotic patients by venous occlusion plethysmography. The brachial artery of the nondominant arm was catheterized, and vasoactive drugs were administered: methacholine 4 microg/min; octreotide 20 microg/h, and octreotide 20 microg/h + methacholine 4 microg/min. Each infusion, lasting 5 minutes, was followed by saline for washout. FBF was measured in both arms during the last minute of each infusion and at the end of washout, with the uninfused arm acting as the control. Nitrates and nitrites, octreotide, and glucagon blood levels were determined at baseline and after each infusion. Percent change in flow (%triangle up) was obtained by comparing the flow during drug administration to that during the preceding saline infusion. Saline infusion did not alter FBF, but octreotide infusion resulted in a 34% +/- 7.7 (P <.005) reduction in FBF in the infused arm. FBF in the control arm was unchanged despite a significant decrease in systemic glucagon levels. Methacholine infusion increased FBF around 300%, which was not altered by the concomitant infusion of octreotide. Octreotide has a local vasoconstrictive effect that seems nitric oxide (NO)-independent. Octreotide probably has a facilitating effect over vasoconstrictors increased in chronic liver diseases.

Diagnosing portal hypertension.

It is important to diagnose portal hypertension owing to its devastating complications. Clinicians need to be able to recognize physical signs and symptoms associated with portal hypertensive states. When in doubt, appropriate diagnostic measures need to be performed and a definite diagnosis made. Hepatic venous pressure gradient (HVPG) is often used as a surrogate measurement of portal pressure. HVPG can be obtained safely and conveniently on an outpatient basis. It can also be used to assess efficacy of various treatment modalities. Knowledge of pathophysiology of portal hypertension has provided the basis for further trials in both novel treatment modalities and diagnostic methods.

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.

NO overproduction by eNOS precedes hyperdynamic splanchnic circulation in portal hypertensive rats.

Chronic high blood flow and the hyperdynamic circulatory syndrome in portal hypertension are associated with endothelial constitutive nitric oxide (NO) synthase (eNOS) upregulation and increased NO release. In portal vein-ligated (PVL) rats the splanchnic circulation is not yet hyperdynamic on day 3 postoperatively. In vitro perfused superior mesenteric arteries (SMAs) of day 3 PVL and sham rats were challenged with increasing flow rates or the alpha-adrenoreceptor agonist methoxamine (30 and 100 microM) before and after incubation with the NO inhibitor, Nomega-nitro-L-arginine (L-NNA, 10(-4) M). Perfusate NO metabolite (NOx) concentrations were measured by chemiluminescence. PVL rats expressed a significant hyporesponsiveness to increases in flow rate or methoxamine that was overcome by incubation with L-NNA. The PVL vasculature showed significantly higher slopes of NOx production vs. flow-induced shear stress, higher increases in perfusate NOx concentration in response to methoxamine, and higher eNOS protein levels (Western blot) compared with sham rats. In conclusion, eNOS-upregulation and increased NO release by the SMA endothelium occur before the development of the hyperdynamic splanchnic circulation, suggesting a primary role of NO in the pathogenesis of arterial vasodilatation.

Low-dose midazolam sedation: an option for patients undergoing serial hepatic venous pressure measurements.

The hepatic venous pressure gradient (HVPG) is becoming increasingly used clinically. It is useful in the differential diagnosis of portal hypertension and provides a prognostic index in cirrhotic patients. Performance of serial measurements has been shown to be useful in guiding pharmacological therapy of portal hypertension and variceal hemorrhage. The technique is safe to perform; however, many patients are anxious and reluctant to undergo serial measurements. The effects of sedatives on portal pressure measurements have not yet been defined. The objective of this study was to evaluate the effects of midazolam on the HVPG. Twenty patients with compensated cirrhosis were included in this prospective, double-blind study. The HVPG was determined by subtracting the free hepatic venous pressure (FHVP) from the wedged hepatic venous pressure (WHVP). Patients were randomized to receive either placebo, 0.02 mg/kg midazolam, or 0.03 mg/kg midazolam, administered intravenously over 3 minutes. Immediately after drug administration and every 3 minutes thereafter, for a total of 30 or 40 minutes, measurements were repeated. Three hours later, patients were asked to state whether the sedative affected their state of comfort/relaxation. The effects of both doses of midazolam on HVPG did not differ significantly from those of placebo. Furthermore, neither dose of midazolam induced significant changes in HVPG as compared with baseline values. However, higher-dose midazolam (0.03 mg/kg) was associated with significant reductions in FHVP from baseline and a tendency for a reduction in WHVP. Both doses significantly increased patient comfort and relaxation during the test. Midazolam, used at a dose of 0.02 mg/kg, is effective in increasing patient comfort and relaxation during hepatic venous pressure measurements, without significantly affecting pressures (HVPG, WHVP, or FHVP). It is therefore an acceptable option for patients undergoing serial hepatic venous pressure measurements.

Adrenomedullin, a vasodilator peptide implicated in hemodynamic alterations of liver cirrhosis: relationship to nitric oxide.

This prospective cohort study was aimed at investigating the role of adrenomedullin, a potent vasodilator peptide, in liver cirrhosis and its relationship with nitric oxide and cytokines. Overall, 66 consecutive patients with liver cirrhosis and 15 controls matched for age and sex distribution were included. Adrenomedullin levels in patients with cirrhosis were higher than in controls [28.1 (23.5-34.8) vs 21.9 (21.1-26.4) pmol/liter, P = 0.002]. Child class A patients had adrenomedullin levels similar to those of controls, but lower than patients in class B and C, respectively (P = 0.01). Patients with ascites showed more elevated adrenomedullin levels than patients without (P = 0.001). Adrenomedullin levels had significant correlations with aldosterone (r = 0.55; P < 0.001), plasma renin activity (r = 0.49; P < 0.001) and nitrates-nitrites levels (r = 0.52; P < 0.001). Weak correlations were found with tumor necrosis factor-alpha and interleukin-6. This study shows that high levels of adrenomedullin in liver cirrhosis correlate with features associated with plasma volume expansion, and suggests that, in late stages of cirrhosis, adrenomedullin might contribute to vasodilatation by increasing the generation of nitric oxide.

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.

Nitric oxide and portal hypertension: its role in the regulation of intrahepatic and splanchnic vascular resistance.

In recent years it has become well established that nitric oxide (NO) plays a crucial role in the hemodynamic abnormalities that develop in chronic portal hypertension. The purpose of this review is to summarize the available data and current concepts regarding the involvement of NO in the pathophysiologic changes in the micro-circulation of the liver and the splanchnic and systemic circulation that associate portal hypertension.

Enhanced release of nitric oxide in response to changes in flow and shear stress in the superior mesenteric arteries of portal hypertensive rats.

Increased nitric oxide (NO) release has been implicated in the pathogenesis of the hyperdynamic circulation in portal hypertension. NOS 3 (eNOS) causes NO release from the endothelium in response to physical stimuli, such as increased blood flow and shear stress. We evaluated the functional activity of the endothelium in the superior mesenteric arterial bed of portal hypertensive rats through direct measurement of NO metabolites (NOx) during changes in flow and shear stress. The in vitro perfusion system (McGregor) was used in sham and portal vein-ligated (PVL) rats. Shear stress was applied gradually to superior mesenteric arterial beds by increasing the perfusion rate. Flow studies were performed serially before and after incubation with either Krebs solution alone or with the NO-inhibitor, NG-monomethyl-L-arginine (L-NMMA). NOx concentrations in the perfusate were measured using chemiluminescence. The slope of NOx release versus flow-induced shear stress was calculated. Before L-NMMA administration, NOx concentrations and release of NO in PVL rats were significantly elevated in comparison with sham rats at flow rates of 32, 40, and 48 mL/min. The slope of NOx production versus shear stress index was significantly higher in PVL than in sham rats. After L-NMMA incubation, the decrease in slope was significantly larger in PVL rats. This study provides direct evidences of an increased NO synthesis by the superior mesenteric arterial vascular endothelium of PVL animals in response to shear stress. The increased NO output in response to shear stress suggests an adaptative mechanism developed by the vascular endothelial cells in response to a chronic increase in flow-mediated shear stress.

Endothelial dysfunction and decreased production of nitric oxide in the intrahepatic microcirculation of cirrhotic rats.

Increased intrahepatic resistance in cirrhotic livers is in part caused by increased vascular tone. Several morphological abnormalities have been described in the sinusoidal endothelial cells of cirrhotic livers, but the functional impact of these abnormalities on the intrahepatic vascular tone has not been studied. The aim of this study was to investigate the intrahepatic endothelial function and the role of nitric oxide (NO) with regard to vascular tone in cirrhotic livers. Isolated rat liver perfusions were performed in cirrhotic rats (induced by chronic carbon tetrachloride inhalation) and weight-matched normal controls. After preconstricting the intrahepatic microcirculation with methoxamine (10(-4) mol/L), response to cumulative doses of receptor-mediated endothelial agonist, acetylcholine (10(-7) mol/L-10(-5) mol/L), was obtained. In another series, response to the receptor-independent endothelial agonist, calcium ionophore A23187 (10(-7) mol/L and 3 x 10(-7) mol/L), was obtained in the absence and presence of Nomega-nitro-L-arginine (NNA) and indomethacin. In a third series of rats, nitrate and nitrite production was measured in the perfusate of perfused normal and cirrhotic livers. There was significantly less vasorelaxation in cirrhotic livers as compared with normal livers in response to acetylcholine and calcium ionophore A23187 (P < .0001). The impaired vasorelaxation was a result of a decrease in both NO-mediated and non-NO-mediated components of vasorelaxation. Cirrhotic livers from ascitic rats had significantly less vasorelaxation as compared with livers from nonascitic rats (P < .005). There was significantly less production of nitrates and nitrites in cirrhotic livers (P < .05). The liver microcirculation of cirrhotic livers is characterized by endothelial dysfunction that results in impaired release of endothelial relaxing factors including NO.

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.