Simvastatin Enhances Hepatic Nitric Oxide Production and Decreases the Hepatic Vascular Tone in Patients With Cirrhosis

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1 GASTROENTEROLOGY 2004;126: Simvastatin Enhances Hepatic Nitric Oxide Production and Decreases the Hepatic Vascular Tone in Patients With Cirrhosis CARMEN ZAFRA, JUAN G. ABRALDES, JUAN TURNES, ANNALISA BERZIGOTTI, MERCEDES FERNÁNDEZ, JUAN C. GARCÍA-PAGÁN, JUAN RODÉS, and JAIME BOSCH Hepatic Hemodynamic Laboratory, Liver Unit, Institut de Malalties Digestives, Hospital Clinic, Institut d Investigacions Biomediques August Pi i Sunyer, Hospital Clínic, University of Barcelona, Barcelona, Spain Background & Aims: In cirrhosis, an insufficient release of nitric oxide contributes to increased hepatic resistance and portal pressure and enhances the postprandial increase in portal pressure. We hypothesized that simvastatin, which enhances Akt-dependent endothelial nitric oxide synthase phosphorylation, may increase hepatic nitric oxide release and decrease hepatic resistance in patients with cirrhosis and portal hypertension. Methods: In protocol 1, 13 patients had measurements of the hepatic venous pressure gradient, hepatic blood flow, mean arterial pressure, cardiac output, and nitric oxide products before and 30 and 60 minutes after 40 mg of simvastatin. In protocol 2, 17 patients were randomized to receive placebo or simvastatin (40 mg) 12 hours and 1 hour before the study. After baseline measurements of the hepatic venous pressure gradient, hepatic blood flow, and nitric oxide products, a standard liquid meal was given, and measurements were repeated at 15, 30, and 45 minutes. Results: In protocol 1, acute simvastatin did not modify the hepatic venous pressure gradient but increased the hepatic blood flow (21% 13% at 30 minutes; P 0.01) and decreased hepatic sinusoidal resistance by 14% 11% (P 0.04). Nitric oxide product levels significantly increased in hepatic venous blood (from nmol ml 1 to nmol ml 1 ; P 0.04), but not in peripheral blood. Systemic hemodynamics were not modified. In protocol 2, simvastatin pretreatment significantly attenuated the postprandial increase in hepatic venous pressure gradient (mean peak increase, 10% 9% vs. 21% 6% in placebo; P 0.01). Hepatic blood flow increased similarly in the 2 groups. Hepatic nitric oxide products increased in the simvastatin group but not in the placebo group. Conclusions: Simvastatin administration increases the hepatosplanchnic output of nitric oxide products and decreases hepatic resistance in patients with cirrhosis. Portal hypertension is an almost unavoidable consequence of advanced liver disease and is directly or indirectly responsible for major clinical complications such as bleeding from ruptured esophageal varices, a leading cause of death in patients with cirrhosis. Increased hepatic resistance is the first pathophysiological phenomenon that causes portal hypertension in cirrhosis. Although traditionally considered a mechanical consequence of the disruption of the liver vascular architecture caused by the cirrhotic process, an increased hepatic vascular tone significantly contributes to increased hepatic resistance and portal pressure in cirrhosis. 1,2 It has been estimated that this accounts for approximately 30% of the increase in hepatic vascular resistance, 1 and its modulation has become a key therapeutic target. 3 Studies in experimental models of cirrhosis suggest that endothelial nitric oxide release is impaired in liver microvasculature 4 7 and that this may be a major factor that contributes to increased hepatic resistance in cirrhosis. In addition, such insufficient NO availability may explain the incapacity of the intrahepatic vasculature to relax in response to acute increases in portal blood flow, such as those induced by meals. 8 This impaired endothelium-dependent relaxation (endothelial dysfunction) may be further aggravated by postprandial hyperglycemia and hypertriglyceridemia. 9 As a result, in cirrhotic patients, marked postprandial increases in portal pressure occur. 8,10 15 Both unaltered 5,6 and decreased 16 protein levels of endothelial nitric oxide synthase (enos) have been found in cirrhosis, but decreased hepatic enos activity has been uniformly reported. 4 7,17 This has been attributed to complex posttranslational modifications of enos. Indeed, in the cirrhotic liver, enos activity has been shown to be down-regulated because of an increased interaction of enos with the inhibitory protein caveolin-1. 6,7 Therefore, up-regulating enos activity in the Abbreviations used in this paper: enos, endothelial nitric oxide synthase; HBF, hepatic blood flow; HSR, hepatic sinusoidal resistance; HVPG, hepatic venous pressure gradient; MAP, mean arterial pressure; NO, nitric oxide; NOx, nitric oxide products by the American Gastroenterological Association /04/$30.00 doi: /j.gastro

2 750 ZAFRA ET AL. GASTROENTEROLOGY Vol. 126, No. 3 cirrhotic liver may constitute a new strategy to correct the increased hepatic vascular tone in these patients. 18,19 Statins, drugs that inhibit the activity of 3-hydroxyl- 3-methyl coenzyme A reductase, have been shown to decrease the incidence of complications of atherosclerosis. 20 The beneficial effects of statins were attributed to their lipid-lowering effects, but it is now well established that the benefit extends beyond cholesterol reduction. 21 Statins have pleiotropic effects: they decrease oxidative stress and inflammation at the vessel wall, have antithrombotic properties, and improve endothelial function, increasing NO production in endothelial cells This last effect seems to be mediated through the phosphatidylinositol 3-kinase dependent activation of protein kinase Akt, leading to enos phosphorylation at Ser 1177, with subsequent increased activity. 24 We therefore hypothesized that, in patients with cirrhosis, statins could increase NO production in the liver microcirculation, thereby decreasing hepatic resistance. This study tested this hypothesis by examining the effects of simvastatin administration on resting and mealstimulated hepatic hemodynamics and NO output in patients with cirrhosis. Materials and Methods Patients This study was performed in 30 patients with liver cirrhosis referred for the hemodynamic evaluation of portal hypertension: 13 were included in protocol 1 and 17 in protocol 2. The protocols fulfilled the criteria of the Declaration of Helsinki (revision of Edinburgh, 2000) and were approved by the Human Research Committee of the Hospital Clínic of Barcelona, Spain, in May The nature of the study was explained to the patients, and written, informed consent was obtained in each case. All patients had liver cirrhosis diagnosed by clinical, biochemical, ultrasonographic, and/or histological criteria. Patients were considered eligible for the study if they were found to have a hepatic venous pressure gradient (HVPG) 12 mm Hg during the hemodynamic study. Exclusion criteria were pregnancy; advanced liver failure (defined as a prothrombin rate 40% and bilirubin 5 mg/dl, hepatic encephalopathy grade II or higher, or a Child Pugh score 12 points); portal vein thrombosis; hepatocellular carcinoma; cardiac, renal, or respiratory failure; previous surgical or transjugular intrahepatic portosystemic shunt; and insulin-dependent diabetes. Hemodynamic Studies After an overnight fast, patients were transferred to the hepatic hemodynamic laboratory. With the patient under local anesthesia, a double-lumen venous catheter introducer (Axcess; Maxxim Medical, Athens, TX) was placed in the right jugular vein under ultrasonographic guidance by using the Seldinger technique. Under fluoroscopic control, a 5F balloon-tipped catheter (Medi-Tech; Boston Scientific Cork Ltd., Cork, Ireland) was advanced into the main right hepatic vein, where it was kept for the entire study. Through the other lumen, a 5F Swan Ganz catheter (Edwards Laboratory, Los Angeles, CA) was advanced into the pulmonary artery and kept in position until the end of the study. These catheters allowed serial measurements of wedged and free hepatic venous pressures, cardiopulmonary pressures, and cardiac output (thermal dilution). Preceded by a priming dose (5 mg), a solution of indocyanine green (Pulsion Medical Systems, Munich, Germany) was infused intravenously at a constant rate of 0.2 mg/min. After an equilibration period of at least 40 minutes, 4 separate sets of simultaneous samples of peripheral and hepatic venous blood were obtained for the measurement of hepatic blood flow (HBF), as previously described. 29 Mean arterial pressure (MAP) was measured every 5 minutes by an external automatic sphygmomanometer (Marquette Electronics, Milwaukee, WI). Heart rate was derived from continuous electrocardiogram monitoring. All measurements were performed in triplicate in each study period, and permanent tracings were obtained on a multichannel recorder (Marquette Electronics). Portal pressure was estimated from the HVPG: the difference between wedged hepatic venous pressure and free hepatic venous pressure. The hepatic sinusoidal resistance (HSR) was estimated as HVPG 80/HBF (dyne s cm 5 ; HVPG, mm Hg; HBF, ml/min). The systemic vascular resistance index was calculated as MAP RAP 80/CI (dyne s m 2 cm 5 ; MAP and RAP [right atrial pressure], mm Hg). Laboratory Measurements To measure serum NO products (NOx; NO 2 and NO 3 ), samples were ultrafiltered (PL-10 Ultrafree-MC centrifugal filter units; Millipore Corp., Bedford, MA) at 1200g for 1 hour to remove proteins before analysis. Filtered serums were refluxed in glacial acetic acid containing sodium iodide. Under these conditions, NO 2 and NO 3 are reduced to NO, which, after reacting with ozone, can be quantified by a chemiluminescence detector (Nitric Oxide Analyzer, NOA 280; Sievers Instruments, Boulder, CO). Protocol 1. This protocol investigated the effects of a single dose of simvastatin on the hepatic and systemic circulation. The study was performed on 13 portal hypertensive cirrhotics (9 men and 4 women). The clinical data of these patients are given in Table 1. After baseline measurements were obtained, all patients received 40 mg of simvastatin orally, and hemodynamic measurements were repeated at 30 and 60 minutes. Blood samples for NOx measurements were obtained from a peripheral vein and from the hepatic vein at baseline and at 30 minutes. Protocol 2. This protocol, aimed at studying the effects of simvastatin on meal-stimulated flow-mediated vasodilation on hepatic hemodynamics, was performed on 17 patients (14 men and 3 women). The clinical data of these patients are given in Table 1. Patients were randomized to

3 March 2004 SIMVASTATIN FOR PATIENTS WITH CIRRHOSIS 751 Table 1. Baseline Clinical and Laboratory Data From Patients Included in Protocol 1 and 2 Variable Protocol 1: Simvastatin (n 13) Simvastatin (n 9) Protocol 2 Placebo (n 8) Sex (M/F) 9/4 8/1 6/2 Age (y) Child Pugh score Varices (n) History of variceal bleeding (n) Ascites (n) Creatinine (mg/dl) Albumin (g/l) Bilirubin (mg/dl) Platelet count ( 10 9 /L) Prothrombin activity (%) NOTE. Results are expressed as mean SD unless otherwise noted. receive orally in double-blind conditions either placebo (10 mg of lactase; n 8) or simvastatin (40 mg; n 9), 12 hours and 1 hour before the study. The test meal was given 1 hour after the second dose of simvastatin (1) because previous studies have shown persistent effects (for up to 24 hours) of statins on flow-mediated dilation in other vascular beds 30,31 and (2) to avoid any confounding effect on the postprandial hyperemia due to acute increases in HBF early after simvastatin. After baseline measurements were obtained, a standard liquid meal (400 ml) containing 25 g of proteins, 80.8 g of carbohydrates, and g of lipids, for a total of kj (Ensure Plus; Abbott Laboratories B.V., Zwolle, the Netherlands), was administered in 5 minutes. Postprandial hemodynamic measurements were repeated as described at 15, 30, and 45 minutes. 8 HBF and cardiopulmonary pressures were measured only at 30 minutes. Blood samples from a peripheral vein and from the hepatic vein were taken at baseline and at 30 minutes for NOx measurements. Statistics Statistical analyses were performed with the SPSS 10.0 statistical package (SPSS Inc., Chicago, IL). All results are expressed as mean SD. Comparisons within each group were performed with the Student t test for paired data or with the Wilcoxon test, and comparisons between groups were performed with Student t tests for unpaired data. Analysis of variance (ANOVA) for repeated measurements was used when appropriate. The Bonferroni correction was applied for comparisons between each level of the variables and baseline values. Significance was established at a P level of Results Protocol 1 Baseline hemodynamic findings in this series were similar to those observed in previous studies from our laboratory. The oral administration of 40 mg of simvastatin significantly increased HBF (21% 13% at 30 minutes, P 0.02 vs. baseline; 14% 23% at 60 minutes, P 0.3 vs. baseline), without significant changes in the HVPG. Consequently, the estimated HSR was significantly decreased (14% 11% at 30 minutes, P 0.02 vs. baseline; 11% 17% at 60 minutes, P 0.3 vs. baseline). There were no changes in systemic hemodynamics (Table 2). Simvastatin administration significantly increased hepatic vein NOx (from nmol ml 1 to nmol ml 1 ; P 0.04). Peripheral levels of NOx were not significantly modified (from nmol ml 1 to nmol ml 1 ; P 0.19). Protocol 2 There were no significant differences in baseline splanchnic and systemic hemodynamics between patients randomized to receive simvastatin or placebo, although the baseline HVPG was slightly lower in the placebo group (Table 3). Patients in the placebo group (n 8) showed a brisk, statistically significant postprandial increase in HVPG (ANOVA; P 0.01) that was already present at 15 minutes and was maintained during the entire 45-minute observation period (Table 3; Figure 1). The increase in HVPG was associated with an increase in HBF (Table 3). After the test meal, there were no significant changes in MAP or heart rate (Table 3). In patients who received simvastatin (n 9), HVPG also increased significantly after the meal (ANOVA; P 0.04; Table 3). However, the magnitude of the HVPG increase was less than half of that observed in the placebotreated patients ( mm Hg vs mm Hg at 15 minutes; mm Hg vs mm Hg at 30 minutes; and mm Hg vs mm Hg at 45 minutes; P 0.03). Because of the lower

4 752 ZAFRA ET AL. GASTROENTEROLOGY Vol. 126, No. 3 Table 2. Protocol 1: Systemic and Splanchnic Hemodynamic Changes After Simvastatin Administration Variable Baseline 30 min 60 min P value (ANOVA) WHVP (mm Hg) NS FHVP (mm Hg) NS HVPG (mm Hg) NS MAP (mm Hg) NS HR (bpm) NS HBF (ml/min) a HSR (dyne s cm 5 ) a SVRI (dyne s cm 5 m 2 ) NS CI (L m 2 min 1 ) NS Hepatic NOx (nmol ml 1 ) Peripheral NOx (nmol ml 1 ) NS WHVP, wedged hepatic venous pressure; FHVP, free hepatic venous pressure; HVPG, hepatic venous pressure gradient; MAP, mean arterial pressure; HR, heart rate; bpm, beats per minute; HBF, hepatic blood flow; HSR, hepatic vascular resistance; SVRI, systemic vascular resistance index; CI, cardiac index; NOx, nitric oxide products; ANOVA, analysis of variance; NS, not significant. a P 0.05 vs. baseline. baseline HVPG in the placebo group, absolute values after the meal were not different from those of simvastatin-treated patients (Table 3). The attenuation of the postprandial increase in HVPG occurred despite a similar increase in HBF in patients who received simvastatin or placebo (Table 3; Figure 1). This was due to the significant reduction in HSR in the simvastatin group but not in the placebo group ( dyne s m 2 cm 5 vs dyne s m 2 cm 5 ; P 0.03; Figure 1). Hepatic NOx levels increased significantly after the meal in patients who received simvastatin (from nmol ml 1 to nmol ml 1 ; P 0.05), but not in patients who received placebo (Table 3). Peripheral levels were not significantly modified. Discussion Increases in hepatic resistance in cirrhosis occur predominantly through mechanical factors, but there is also a vasculogenic component that is clearly reversible. 19 This concept was first shown by Bhathal and Groszmann 1 in the isolated perfused cirrhotic rat liver. The vasoregulatory pathways involved in the increased hepatic vascular tone of the cirrhotic liver include the NO/guanosine 3,5 -cyclic monophosphate system, endothelins, the sympathetic -adrenergic pathway, thromboxane A 2, leukotrienes, and the renin angiotensin system. 4,5,32 35 Insufficient NO production by endothelial cells in the liver microvasculature seems to be the major factor involved in increased hepatic resistance in cirrhosis. 4 6,17,19 It is implicit that delivering NO to the liver circulation should result in a decrease in hepatic resistance. A straightforward way to achieve this goal is to generate bioavailable NO that diffuses directly to the effector contractile cells by means of NO donors. This is the case with organic nitrates such as isosorbide-5-mononitrate, which have been used in cirrhosis for more than a decade. 36 Although these agents are effective in reducing Table 3. Protocol 2: Postprandial Changes in Systemic and Splanchnic Hemodynamics After Simvastatin and Placebo Simvastatin Placebo Variable Baseline 15 min 30 min 45 min P value (ANOVA) Baseline 15 min 30 min 45 min P value (ANOVA) WHVP (mm Hg) a a a a a a FHVP (mm Hg) NS NS HVPG (mm Hg) a a a a a 0.01 HBF (ml/min) HSR (dyne s cm 5 ) NS MAP (mm Hg) NS NS HR (bpm) NS NS CI (L m 2 min 1 ) NS NS SVRI (dyne s m 2 cm 5 ) NS NS Hepatic NOx (nmol ml 1 ) NS Peripheral NOx (nmol ml 1 ) NS NS NOTE. Results are expressed as mean SD. WHVP, wedged hepatic venous pressure; FHVP, free hepatic venous pressure; HVPG, hepatic venous pressure gradient; MAP, mean arterial pressure; HR, heart rate; HBF, hepatic blood flow; HSR, hepatic sinusoidal resistance; SVRI, systemic vascular resistance index; CI, cardiac index; NOx, nitric oxide products; ANOVA, analysis of variance; NS, not significant. a P 0.05 vs. baseline.

5 March 2004 SIMVASTATIN FOR PATIENTS WITH CIRRHOSIS 753 Figure 1. Protocol 2: absolute postprandial changes observed 30 minutes after the test meal in hepatic venous pressure gradient (HVPG), hepatic blood flow (HBF), and hepatic sinusoidal resistance (HSR) in patients pretreated with simvastatin (black bars) or placebo (white bars) (error bars represent SEM). portal pressure, 29,37 their systemic vasodilatory effects could exacerbate the systemic hypotension of cirrhotic patients with adverse effects on renal function. 38 Liverselective NO donors such as V-pyrro/NO 39 or NCX may overcome this problem, but they have not been tested in patients so far. Data from experimental models of cirrhosis indicate that the reduction in NO production may occur despite normal hepatic enos messenger RNA and protein levels 4 7 ; this suggests a failure in posttranslational mechanisms that regulate enos activity. Attempts to increase NOS activity have included the overexpression of enos 41 or neuronal nitric oxide synthase (nnos) 42 by transfecting the liver parenchyma with adenovirus encoding these enzymes or a constitutively active form of Akt. 43 This approach has been successful in decreasing the portal pressure in animal models of cirrhosis, 16,42,43 but it is far from being applicable to patients. A simpler way of modulating the NO/guanosine 3,5 -cyclic monophosphate system would be to increase the catalytic activity of NOS by pharmacological means. It has been recently shown in vitro that the 3-hydroxyl-3-methyl coenzyme A inhibitor simvastatin rapidly induced Akt phosphorylation selectively at endothelial cells, 24 which resulted in increased Akt-dependent enos phosphorylation and ensuing increased endothelial NO production. These effects were evident as early as 15 minutes after simvastatin exposure. Simvastatin has also been shown to decrease the expression of the enos inhibitory protein caveolin-1, 44 whose overexpression in the liver 6 contributes to decrease enos activity and NO production in liver cirrhosis 6,7,45,46 and to stabilize enos messenger RNA, with increased production of enos protein that is not seen before 24 hours. 47 With this background, we postulated that the administration of simvastatin could decrease hepatic resistance in cirrhosis by increasing NO production at hepatic endothelial cells. The results of our study show that, in patients with cirrhosis and portal hypertension, simvastatin acutely increases hepatic vein NO levels and reduces hepatic resistance. However, the decrease in hepatic resistance achieved by simvastatin was not associated with any decrease in portal pressure, because of a concomitant increase in HBF. Because direct, NO-independent vasoactive effects of simvastatin have not been described, the hemodynamic changes observed in our study were most likely mediated by the up-regulation of NO production at the endothelial level. This is supported by the significant increase in plasma NOx selectively at the hepatic vein, which suggests an up-regulation in NO production localized at the hepatosplanchnic territory. Both the hepatic hemodynamic effects and the increase in NO induced by simvastatin were observed as soon as 30 minutes after drug administration, a finding that fits with an effect on posttranslational regulation of enos, rather than transcriptional up-regulation. Hepatic vein NOx levels increased further after a meal in patients pretreated with simvastatin, but not with placebo, which indicates that simvastatin not only increased basal NO production, but also restored a vasorelaxing response after the volume load caused by postprandial hyperemia. 8 As a consequence, the brisk postprandial increase in HVPG was attenuated. Although the acute effects of simvastatin

6 754 ZAFRA ET AL. GASTROENTEROLOGY Vol. 126, No. 3 were short lived (i.e., changes at 60 minutes when the test meal was administered were no longer significant), previous investigations have shown that the effects of a single statin dose on flow-stimulated endothelial-dependent vasodilation may persist for longer periods 30,31 and are maintained or even enhanced during short-term 48,49 or long-term 50,51 continued administration. These findings show that the hepatic vascular bed also shows such persistent flow-mediated dilation. Our findings show no direct therapeutic potential for simvastatin due to the absence of any decrease in portal pressure. Although there are abundant data indicating that strategies based on supplementing NO enhance the effects of -blockers on portal pressure, whether statins might have such an effect remains to be proven. 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Hepatology 2002;36: Received February 20, Accepted December 4, Address requests for reprints to: Jaime Bosch, M.D., Hepatic Hemodynamic Laboratory, Liver Unit, Hospital Clinic, Villarroel 170, Barcelona, Spain. jbosch@medicina.ub.es; fax: (34) Supported in part by grants from the Fondo de Investigación Sanitaria (PI ) (01/9356; J.G.A.) and from the Instituto de Salud Carlos III (C03/02). The authors thank Angels Baringo, Laura Rocabert, and Rosa Sáez for their expert technical assistance.