Evidence Against a Role for NADPH Oxidase Modulating Hepatic Vascular Tone in Cirrhosis
2007; Elsevier BV; Volume: 133; Issue: 3 Linguagem: Inglês
10.1053/j.gastro.2007.06.021
ISSN1528-0012
AutoresJordi Gracia‐Sancho, Bàrbara Laviña, Aina Rodríguez–Vilarrupla, Ralf P. Brandes, Mercedes Fernández, Jaume Bosch, Juan Carlos García–Pagán,
Tópico(s)Liver Disease Diagnosis and Treatment
ResumoBackground & Aims: Increased hepatic vascular resistance in cirrhosis is in part due to reduced nitric oxide (NO) bioavailability. This is related to insufficient NO synthesis from endothelial nitric oxide synthase and to enhanced NO scavenging by superoxide radicals (O2−). Nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase is an important source of O2− that increases vascular tone in different cardiovascular disorders. Thus, our aims were to study the molecular and biochemical state of NADPH-oxidase in cirrhotic livers and to investigate its possible role in modulating hepatic vascular tone in cirrhosis. Methods: NADPH-oxidase expression and enzymatic activity were determined in control (n = 8) and CCl4-cirrhotic (n = 8) rat livers. Additional control (n = 6) and CCl4-cirrhotic (n = 10) rats were treated with apocynin (a selective NADPH-oxidase inhibitor) or its vehicle. Mean arterial pressure, portal pressure, and superior mesenteric arterial blood flow were measured in vivo. Moreover, hepatic endothelial function was evaluated in isolated and perfused rat livers by dose-response curves to acetylcholine. In addition, in 6 control and 6 cirrhotic human livers NADPH-oxidase activity and expression were evaluated. Results: Rat cirrhotic livers had no increased NADPH-oxidase protein expression or activity in relation to control livers. NADPH-oxidase inhibition did not modify splanchnic or systemic hemodynamics in control or cirrhotic rats and did not improve the impaired endothelial-dependent vasodilatory response to acetylcholine of cirrhotic livers. Human cirrhotic livers also did not exhibit increased NADPH-oxidase expression or activity. Conclusions: Our study shows that NADPH-oxidase activity is decreased in the cirrhotic livers and therefore cannot explain increased hepatic O2−, endothelial dysfunction, and increased vascular tone in cirrhotic livers. Background & Aims: Increased hepatic vascular resistance in cirrhosis is in part due to reduced nitric oxide (NO) bioavailability. This is related to insufficient NO synthesis from endothelial nitric oxide synthase and to enhanced NO scavenging by superoxide radicals (O2−). Nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase is an important source of O2− that increases vascular tone in different cardiovascular disorders. Thus, our aims were to study the molecular and biochemical state of NADPH-oxidase in cirrhotic livers and to investigate its possible role in modulating hepatic vascular tone in cirrhosis. Methods: NADPH-oxidase expression and enzymatic activity were determined in control (n = 8) and CCl4-cirrhotic (n = 8) rat livers. Additional control (n = 6) and CCl4-cirrhotic (n = 10) rats were treated with apocynin (a selective NADPH-oxidase inhibitor) or its vehicle. Mean arterial pressure, portal pressure, and superior mesenteric arterial blood flow were measured in vivo. Moreover, hepatic endothelial function was evaluated in isolated and perfused rat livers by dose-response curves to acetylcholine. In addition, in 6 control and 6 cirrhotic human livers NADPH-oxidase activity and expression were evaluated. Results: Rat cirrhotic livers had no increased NADPH-oxidase protein expression or activity in relation to control livers. NADPH-oxidase inhibition did not modify splanchnic or systemic hemodynamics in control or cirrhotic rats and did not improve the impaired endothelial-dependent vasodilatory response to acetylcholine of cirrhotic livers. Human cirrhotic livers also did not exhibit increased NADPH-oxidase expression or activity. Conclusions: Our study shows that NADPH-oxidase activity is decreased in the cirrhotic livers and therefore cannot explain increased hepatic O2−, endothelial dysfunction, and increased vascular tone in cirrhotic livers. In cirrhotic livers, increased resistance to portal blood flow is the primary factor in the pathophysiology of portal hypertension.1Bosch J. Garcia-Pagan J.C. Complications of cirrhosis I. Portal hypertension.J Hepatol. 2000; 32: 141-156Abstract Full Text PDF PubMed Scopus (432) Google Scholar This increased resistance is in part due to changes in the hepatic vascular architecture and to an increase in hepatic vascular tone. Endothelial dysfunction, characterized by an impaired endothelium-dependent response to vasodilators, is considered one of the main mechanisms involved in the increased hepatic vascular tone of cirrhotic livers2Gupta T.K. Toruner M. Chung M.K. et al.Endothelial dysfunction and decreased production of nitric oxide in the intrahepatic microcirculation of cirrhotic rats.Hepatology. 1998; 28: 926-931Crossref PubMed Scopus (315) Google Scholar and has been related to both a reduction in nitric oxide (NO) bioavailability3Van de C.M. Van Pelt J.F. Nevens F. et al.Low NO bioavailability in CCl4 cirrhotic rat livers might result from low NO synthesis combined with decreased superoxide dismutase activity allowing superoxide-mediated NO breakdown: A comparison of two portal hypertensive rat models with healthy controls.Comp Hepatol. 2003; 2: 2Crossref PubMed Scopus (35) Google Scholar and to increased synthesis of cyclooxygenase (COX)-1-derived vasoconstrictor prostanoids.4Graupera M. Garcia-Pagan J.C. Pares M. et al.Cyclooxyenase-1 inhibition corrects endothelial dysfunction in cirrhotic rat livers.J Hepatol. 2003; 39: 515-521Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 5Gracia-Sancho J. Laviña B. Rodriguez-Vilarrupla A. et al.Enhanced vasoconstrictor prostanoid production by sinusoidal endothelial cells increases portal perfussion pressure in cirrhotic rat livers.J Hepatol. 2007; 47: 220-227Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar The reduced NO bioavailability of cirrhotic livers has been attributed to a decrease in endothelial-NO-synthase activity.2Gupta T.K. Toruner M. Chung M.K. et al.Endothelial dysfunction and decreased production of nitric oxide in the intrahepatic microcirculation of cirrhotic rats.Hepatology. 1998; 28: 926-931Crossref PubMed Scopus (315) Google Scholar, 6Rockey D.C. Chung J.J. Reduced nitric oxide production by endothelial cells in cirrhotic rat liver: endothelial dysfunction in portal hypertension.Gastroenterology. 1998; 114: 344-351Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar However, in several vascular disorders, such as hypercholesterolemia, hypertension, diabetes, atherosclerosis, and heart failure, reduced NO bioavailability has also been related to increased scavenging by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase–dependent superoxide production.7Cai H. Harrison D.G. Endothelial dysfunction in cardiovascular diseases: the role of oxidant stress.Circ Res. 2000; 87: 840-844Crossref PubMed Scopus (3272) Google Scholar, 8Hink U. Li H. Mollnau H. et al.Mechanisms underlying endothelial dysfunction in diabetes mellitus.Circ Res. 2001; 88: E14-E22Crossref PubMed Google Scholar, 9Li J.M. Shah A.M. Endothelial cell superoxide generation: regulation and relevance for cardiovascular pathophysiology.Am J Physiol Regul Integr Comp Physiol. 2004; 287: R1014-R1030Crossref PubMed Scopus (679) Google Scholar Recent data from our group have demonstrated that cirrhotic rat livers present high levels of superoxide radical10Gracia-Sancho J. Laviña B. Rodriguez-Vilarrupla A. et al.Oxidative stress reduces nitric oxide biodisponibility and may contribute to endothelial dysfunction of cirrhotic livers.J Hepatol. 2006; 44: S75Abstract Full Text PDF Google Scholar and that antioxidants improve flow-mediated vasorelaxation of the hepatic vasculature in patients with cirrhosis.11Hernandez-Guerra M. Garcia-Pagan J.C. Turnes J. et al.Ascorbic acid improves the intrahepatic endothelial dysfunction of patients with cirrhosis and portal hypertension.Hepatology. 2006; 43: 485-491Crossref PubMed Scopus (99) Google Scholar Moreover, an important role for NADPH oxidase has been hypothesized in different liver pathologies, including early alcohol-induced hepatitis, hepatic fibrosis, nonalcoholic fatty liver disease, and ischemia-reperfusion injury.12Kono H. Rusyn I. Yin M. et al.NADPH oxidase–derived free radicals are key oxidants in alcohol-induced liver disease.J Clin Invest. 2000; 106: 867-872Crossref PubMed Scopus (435) Google Scholar, 13Bataller R. Schwabe R.F. Choi Y.H. et al.NADPH oxidase signal transduces angiotensin II in hepatic stellate cells and is critical in hepatic fibrosis.J Clin Invest. 2003; 112: 1383-1394Crossref PubMed Scopus (508) Google Scholar, 14Carmiel-Haggai M. Cederbaum A.I. Nieto N. A high-fat diet leads to the progression of non-alcoholic fatty liver disease in obese rats.FASEB J. 2005; 19: 136-138Crossref PubMed Scopus (274) Google Scholar, 15Harada H. Hines I.N. Flores S. et al.Role of NADPH oxidase–derived superoxide in reduced size liver ischemia and reperfusion injury.Arch Biochem Biophys. 2004; 423: 103-108Crossref PubMed Scopus (56) Google Scholar The current study was aimed at characterizing the molecular and biochemical state of NADPH oxidase in cirrhotic livers and investigating its possible role in modulating hepatic vascular tone. Male Wistar rats weighing 175–200 g underwent inhalation exposure to CCl4 and received phenobarbital in the drinking water as previously described.16Graupera M. Garcia-Pagan J.C. Abraldes J.G. et al.Cyclooxygenase-derived products modulate the increased intrahepatic resistance of cirrhotic rat livers.Hepatology. 2003; 37: 172-181Crossref PubMed Scopus (119) Google Scholar Once the cirrhotic rats developed ascites, administration of CCl4 and phenobarbital was stopped and experimental protocols were started 1 week later. Control animals received only phenobarbital. The animals were kept in environmentally controlled animal facilities at the Institut d'Investigacions Biomèdiques August Pi i Sunyer. Here and in the Hospital Clínic all experiments were performed according to the criteria of the Committee for the Care and Use of Laboratory Animals. Animals were treated for 7 days with the selective NADPH oxidase inhibitor Apocynin (1.5 mmol/L added to the drinking water; Sigma, Tres Cantos, Madrid, Spain; control rats, n = 6; cirrhotic rats, n = 10) or its vehicle (control rats, n = 6; cirrhotic rats, n = 10). Previous studies have shown that this dose of apocynin and the route of administration is effective at inhibiting NADPH oxidase activity in rats.17Cotter M.A. Cameron N.E. Effect of the NAD(P)H oxidase inhibitor, apocynin, on peripheral nerve perfusion and function in diabetic rats.Life Sci. 2003; 73: 1813-1824Crossref PubMed Scopus (109) Google Scholar, 18Beswick R.A. Dorrance A.M. Leite R. et al.NADH/NADPH oxidase and enhanced superoxide production in the mineralocorticoid hypertensive rat.Hypertension. 2001; 38: 1107-1111Crossref PubMed Scopus (312) Google Scholar, 19Hu L. Zhang Y. Lim P.S. et al.Apocynin but not l-arginine prevents and reverses dexamethasone-induced hypertension in the rat.Am J Hypertens. 2006; 19: 413-418Crossref PubMed Scopus (68) Google Scholar Under anesthesia with intraperitoneal ketamine hydrochloride (100 mg/kg body weight Ketalar; Parke-Davis S.L. El Prat de Llobregat, Barcelona, Spain) and xylazine hydrochloride (5 mg/kg body weight; Sigma), a tracheotomy was performed and a polyethylene PE-240 tubing was inserted into the trachea to ensure a patent airway. Catheters (PE-50) were introduced into the femoral artery to record arterial pressure (mm Hg) and into the portal vein through an ileocolic vein to measure portal pressure (mm Hg). The superior mesenteric artery was then carefully dissected free from connective tissue and a nonconstrictive perivascular transit-time ultrasonic flow probe (1PR, 1 mm diameter; Transonic Systems, Ithaca, NY) was placed around the vessel close to its aortic origin. The flow probe was connected to a flow meter to measure the superior mesenteric artery blood flow (SMABF; mL · min−1 · 100 g−1).20Fernandez M. Garcia-Pagan J.C. Casadevall M. et al.Acute and chronic cyclooxygenase blockage in portal-hypertensive rats: influence in nitric oxide biosynthesis.Gastroenterology. 1996; 110: 1529-1535Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar Resistance in the superior mesenteric artery (mm Hg · mL−1 · min−1 · 100 g−1) was calculated as: (mean arterial pressure − portal pressure)/superior mesenteric artery blood flow. Blood pressures and flows were registered on a multichannel computer-based recorder (PowerLab; ADInstruments, Colorado Springs, CO). The external zero reference point was placed at the midportion of the animal. Hemodynamic data was collected after a 30-minute stabilization period. In a subgroup of animals livers were isolated and perfused by a flow-controlled perfusion system as described previously.21Graupera M. Garcia-Pagan J.C. Titos E. et al.5-Lipoxygenase inhibition reduces intrahepatic vascular resistance of cirrhotic rat livers: a possible role of cysteinyl-leukotrienes.Gastroenterology. 2002; 122: 387-393Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar Briefly, livers were perfused with Krebs buffer in a recirculation fashion with a total volume of 100 mL at a constant flow rate of 35 mL/min. An ultrasonic flow probe (model T201; Transonic Systems) and a pressure transducer were placed on line, immediately ahead of the portal inlet cannula, to continuously monitor portal flow and perfusion pressure. Another pressure transducer was placed immediately after the thoracic vena cava outlet for measurement of outflow pressure. The flow probe and the two pressure transducers were connected to a PowerLab (4SP) linked to a computer using the Chart version 5.0.1 for Windows software (ADInstruments, Mountain View, LA). The average portal flow and inflow and outflow pressures were continuously sampled, recorded, and afterward analyzed. The perfused rat liver preparation was allowed to stabilize for 30 minutes before the studied substances were added. The gross appearance of the liver, stable perfusion pressure, and a stable buffer pH (7.4 ± 0.1) were measured during this period. If any viability criteria were not satisfied, the experiment was discarded. The intrahepatic microcirculation was preconstricted by adding to the reservoir, during a 3-minute period, the α1-adrenergic agonist methoxamine to achieve the final concentration of 10−4 mol/L. Five minutes later, concentration-effect curves to cumulative doses of acetylcholine (Ach; 10−8, 10−7, 10−6 mol/L) were evaluated in control (n = 8) and cirrhotic (n = 12) livers treated with vehicle (n = 10) or apocynin (n = 10). The concentration of Ach was increased by one log unit every 1.5 minutes. Response to cumulative doses of Ach was calculated as percent change in perfusion pressure as previously described.2Gupta T.K. Toruner M. Chung M.K. et al.Endothelial dysfunction and decreased production of nitric oxide in the intrahepatic microcirculation of cirrhotic rats.Hepatology. 1998; 28: 926-931Crossref PubMed Scopus (315) Google Scholar, 21Graupera M. Garcia-Pagan J.C. Titos E. et al.5-Lipoxygenase inhibition reduces intrahepatic vascular resistance of cirrhotic rat livers: a possible role of cysteinyl-leukotrienes.Gastroenterology. 2002; 122: 387-393Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar NADPH oxidase-dependent superoxide anion production, was assessed in control (n = 9) and cirrhotic (n = 9) rat livers by lucigenin-enhanced chemiluminescence.22Brandes R.P. Koddenberg G. Gwinner W. et al.Role of increased production of superoxide anions by NAD(P)H oxidase and xanthine oxidase in prolonged endotoxemia.Hypertension. 1999; 33: 1243-1249Crossref PubMed Scopus (114) Google Scholar, 23Janiszewski M. Souza H.P. Liu X. et al.Overestimation of NADH-driven vascular oxidase activity due to lucigenin artifacts.Free Radic Biol Med. 2002; 32: 446-453Crossref PubMed Scopus (63) Google Scholar Briefly, tissues were excised, immediately snap-frozen in liquid nitrogen, and stored at −80°C for analysis. Tissues were minced thoroughly with mortar under liquid nitrogen. A 10% homogenate was prepared by homogenizing the obtained powder in 1 mL Krebs–hydroxyethyl-piperazine ethanesulafonic acid buffer, containing 0.01 mol/L EDTA and 0.01 mol/L ethylene glycol tetraacetic acid (pH 7.4) (Sigma), by use of a glass-to-glass homogenizer. The homogenate was centrifuged at 1000 g for 10 minutes, to remove unbroken cells and debris. Protein quantification was performed by the Lowry method and the final concentration adjusted to 10 μg/mL. For the chemiluminescence assay, 100 μL aliquots were added to 400 μL of a Krebs–hydroxyethyl-piperazine ethanesulafonic acid assay solution containing lucigenin (5 μmol/L; Sigma) as the electron acceptor. After equilibration and background counts, NADPH (0.1 mmol/L; Sigma) was added as the substrate, and the luminescence counts (relative light units) were monitored continuously over a 3-minute period in a luminometer (Lumat LB 9507, Berthold Technologies, GmbH & Co. KG, Bad Wildbad, Germany), at 37°C. Then, superoxide dismutase (400 U/mL; Sigma) was added and counts were measured again over a 3-minute period. In additional experiments, apocynin (100 μmol/L) was added in the assay mixture before NADPH addition, luminescence counts were measured as described above. Total RNA was isolated from frozen control (n = 8) or cirrhotic (n = 8) livers using the Trizol method (Invitrogen, El Prat de Llobregat, Barcelona, Spain). The RNA was treated with DNAse (Ambion, Austin TX) to eliminate contaminating DNA. For cDNA synthesis, 1 μg of total RNA was retrotranscribed using MLV reverse transcriptase (RT) and random hexamers, as described by the manufacturer (Invitrogen). cDNA templates were amplified by reverse transcriptase–polymerase chain reaction using the fluorescent TaqMan technology (Applied Biosystems, Foster City, CA) on an ABI Prism 7900 sequence Detection System (Applied Biosystems). The probes and primers for the quantification of rat nonphagocytic NADPH oxidase (NOX)2, rat p22phox, and rat p67phox were designed using Primer Express software (Applied Biosystems), whereas the quantification of rat NOX1, rat NOX4, rat p47phox, and the endogenous control 18S RNA was performed using predesigned Gene-Expression-Assays obtained from Applied Biosystems according to the manufacturer's protocol. Each polymerase chain reaction was carried out with 2 μL of the hepatic cDNA sample, 1X TaqMan Universal PCR Master Mix (Applied Biosystems), and primers and probe in a final volume of 20 μL, as recommended by the manufacturer. After an initial denaturation step at 95°C for 10 minutes, 40 cycles were performed as follows: 95°C for 15 seconds and 60°C for 1 minute. All experiments were performed in duplicate, and several negative controls were included. NADPH oxidase subunits expression was related to a standard curve derived from serial dilutions (10−1–10−4) of a random sample cDNA. Standard curves were constructed by plotting the log of standard dilutions versus the threshold cycle (CT) values, CT being the fractional cycle number at which the fluorescence passes a fixed threshold. The mRNA concentration of each NADPH oxidase subunit in hepatic samples was calculated referring the sample CT to the standard curve and normalized with the corresponding value of endogenous control CT as recommended in the TaqMan user's manual. Values were expressed as relative units. Protein expression for NOX2, NOX4, and p22phox in livers from 6 cirrhotic and 6 control rats was assessed by Western blot (the 3 NADPH oxidase subunits for which there are commercially available antibodies for tissue blotting) as follows. Livers were collected, snap frozen in liquid N2, and stored at −80°C until analyzed. Livers were minced thoroughly with mortar and pestle under liquid nitrogen. For each sample, a similar amount of obtained powder were collected in 200 μL triton-lysis buffer containing Tris/HCl (pH 7.4, 20 mmol/L), NaCl (150 mmol/L), NaF (20 mmol/L), Na4P2O7 (10 mmol/L), okadaic acid (10 nmol/L), Na3VO4 (2 mmol/L), antipain (2 μg/mL), aprotinin (2 μg/mL), chymostatin (2 μg/mL), leupeptin (2 μg/mL), pepstatin (2 μg/mL), trypsin inhibitor (2 μg/mL), phenylmethylsulfonylfluoride (40 μg/mL), and TritonX-100 (1% vol/vol), left on ice for 10 minutes, and then centrifuged at 10000 g for 10 minutes. Protein concentration was assessed by the Bradford method. Aliquots from each sample, containing equal amounts of protein (100 μg), were run on a 10% sodium dodecyl sulfate–polyacrylamide gel and transferred to a nitrocellulose membrane. The efficiency of the transfer was visualized by Ponceau staining. The blots were subsequently blocked at room temperature for 1 hour with phosphate-buffered saline containing 0.1% (vol/vol) Tween 20, 5% (wt/vol) bovine serum albumin, and subsequently incubated with primary antibodies overnight at 4°C. Then, membranes were incubated with the appropriate horseradish peroxidase–conjugated secondary antibodies for 1 hour at room temperature. Blots were revealed by chemiluminiscence. Protein expression was determined by densitometric analysis using the Science Lab Image Gauge; images were obtained using Science Lab 2001 Image Gauge (Fuji Photo Film GMBH, Düsseldorf, Germany). After stripping, blots were assayed for glyceraldehyde-3-phosphate dehydrogenase). Quantitative densitometric values of each NADPH oxidase subunit were normalized to glyceraldehyde-3-phosphate dehydrogenase and displayed in histograms. NADPH oxidase activity and NOX2 and NOX4 protein expression were determined, as described previously, in liver tissue specimens from 6 patients with cirrhosis (3 men and 3 women, aged from 44 to 63 years) who received orthotopic liver transplantation for end-stage liver disease between May 2004 and October 2004 (3 alcoholic and 3 postviral cirrhosis). As controls, biopsy specimens were obtained from nontumoral, normal liver tissue from hepatectomy specimens from 6 patients (3 men and 3 woman, aged from 29 to 78 years) who underwent partial liver resection surgery for different reasons (1 patient with focal nodular hyperplasia, metastatic adenocarcinoma of intestinal origin in 3 cases and resection of hepatocarcinoma over normal liver in 2 patients). The study was approved by the Ethical Committee of the Hospital Clinic i Provincial de Barcelona. Methoxamine and other chemical reagents were purchased from Sigma. Statistical analysis was performed using the SPSS 10.0 for Windows statistical package (SPSS Inc., Chicago, IL). The unpaired Student's t test and analysis of variance were used as adequate. All data are reported as means ± SD. Differences were considered significant at a P value <.05. Control and cirrhotic rat livers expressed NOX2, NOX4, p22phox, p47phox, and p67phox mRNA but not NOX1 mRNA. The levels of NOX2, p22phox, p47phox, and p67phox mRNA were significantly increased, but NOX4 mRNA levels were significantly reduced in cirrhotic in comparison with control livers (Figure 1). By contrast, expression of the mRNA NOX1 subunit was almost undetectable, both in control and in cirrhotic livers. Indeed, the expression was about 200 times lower than those observed in mRNA from colon tissue used as positive control (data not shown). Protein expression of both NOX2 and NOX4 was significantly decreased in cirrhotic in comparison with control rat livers (Figure 2). We were unable to detect expression of p22phox protein. NADPH oxidase–dependent superoxide generation, measured by lucigenin-enhanced chemiluminiscence, was not increased, but significantly reduced, in cirrhotic in comparison with control rat livers (Figure 3). Cirrhotic rats had significantly higher portal pressure and superior mesenteric artery blood flow and significantly lower superior mesenteric artery resistance than control rats, without significant differences in mean arterial pressure (Table 1). Treatment with apocynin did not significantly modify any splanchnic or systemic hemodynamic parameters either in control or in cirrhotic rats (Table 1).Table 1Effects of NADPH Oxidase Inhibition by Apocynin Treatment (1.5 mmol/L, During 7 Days) on MAP, PP, SMABF, and SMAR in CT and CH RatsCT-Veh (n = 6)CT-Apo (n = 10)CH-Veh (n = 6)CH-Apo (n = 10)Body wt (g)455 ± 57416 ± 48422 ± 75393 ± 51Liver wt (g)12.7 ± 1.911.8 ± 2.111.1 ± 1.312.1 ± 1.8MAP (mm Hg)112 ± 18110 ± 11105 ± 18107 ± 10PP (mm Hg)8.9 ± 1.28.0 ± 1.913.7 ± 2.5aP < .05 versus CT-Veh.14.0 ± 3.2SMABF (100 g)2.2 ± 0.42.8 ± 0.73.8 ± 1.2aP < .05 versus CT-Veh.3.9 ± 1.3SMAR47.6 ± 12.639.4 ± 14.826.9 ± 16.9aP < .05 versus CT-Veh.22.2 ± 12.7Results are shown as mean ± SD.MAP, mean arterial pressure; PP, portal pressure; SMABF, superior mesenteric artery blood flow; SMAR, superior mesenteric artery resistance; CT, control; CH, CCI4-cirrhotic.a P < .05 versus CT-Veh. Open table in a new tab Results are shown as mean ± SD. MAP, mean arterial pressure; PP, portal pressure; SMABF, superior mesenteric artery blood flow; SMAR, superior mesenteric artery resistance; CT, control; CH, CCI4-cirrhotic. Cirrhotic livers had a significantly greater baseline portal perfusion pressure (9.4 ± 3.1 vs 6.6 ± 2.0 mm Hg; P = .03) and intrahepatic vascular resistance (2.8 ± 0.8 vs 2.0 ± 0.6 mm Hg · g · min/mL; P = .01) than control livers. As expected, control livers showed a dose-dependent vasorelaxation to cumulative doses of Ach. However, cirrhotic livers exhibited endothelial dysfunction as shown by the impaired vasodilatory response to Ach: 10−8 (−6.8 ± 3% vs −15.4 ± 8% in controls; P < .05), 10−7 (−15.3 ± 8% vs −29.6 ± 9%; P = .05), 10−6 mol/L (−19.7 ± 3% vs −49.1 ± 12%; P = .004) (Figure 4). Treatment with apocynin did not significantly modify baseline portal perfusion pressure or the dose response to Ach either in control or in cirrhotic perfused rat livers (Figure 4). NOX4 protein expression was significantly reduced in cirrhotic in comparison with control human livers, whereas no significant differences were observed in NOX2 protein expression and NADPH oxidase enzymatic activity (Figure 5). Increased hepatic vascular resistance in cirrhosis has been related to reduced NO bioavailability within the cirrhotic liver,2Gupta T.K. Toruner M. Chung M.K. et al.Endothelial dysfunction and decreased production of nitric oxide in the intrahepatic microcirculation of cirrhotic rats.Hepatology. 1998; 28: 926-931Crossref PubMed Scopus (315) Google Scholar, 6Rockey D.C. Chung J.J. Reduced nitric oxide production by endothelial cells in cirrhotic rat liver: endothelial dysfunction in portal hypertension.Gastroenterology. 1998; 114: 344-351Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar, 24Matei V. Rodriguez-Vilarrupla A. Deulofeu R. et al.The eNOS cofactor tetrahydrobiopterin improves endothelial dysfunction in livers of rats with CCl4 cirrhosis.Hepatology. 2006; 44: 44-52Crossref PubMed Scopus (91) Google Scholar mainly attributed to reduced endothelial nitric oxide synthase activity.2Gupta T.K. Toruner M. Chung M.K. et al.Endothelial dysfunction and decreased production of nitric oxide in the intrahepatic microcirculation of cirrhotic rats.Hepatology. 1998; 28: 926-931Crossref PubMed Scopus (315) Google Scholar, 6Rockey D.C. Chung J.J. Reduced nitric oxide production by endothelial cells in cirrhotic rat liver: endothelial dysfunction in portal hypertension.Gastroenterology. 1998; 114: 344-351Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar, 24Matei V. Rodriguez-Vilarrupla A. Deulofeu R. et al.The eNOS cofactor tetrahydrobiopterin improves endothelial dysfunction in livers of rats with CCl4 cirrhosis.Hepatology. 2006; 44: 44-52Crossref PubMed Scopus (91) Google Scholar, 25Shah V. Toruner M. Haddad F. et al.Impaired endothelial nitric oxide synthase activity associated with enhanced caveolin binding in experimental cirrhosis in the rat.Gastroenterology. 1999; 117: 1222-1228Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar It has been recently proposed that, in analogy with other vascular disorders,7Cai H. Harrison D.G. Endothelial dysfunction in cardiovascular diseases: the role of oxidant stress.Circ Res. 2000; 87: 840-844Crossref PubMed Scopus (3272) Google Scholar, 8Hink U. Li H. Mollnau H. et al.Mechanisms underlying endothelial dysfunction in diabetes mellitus.Circ Res. 2001; 88: E14-E22Crossref PubMed Google Scholar, 9Li J.M. Shah A.M. Endothelial cell superoxide generation: regulation and relevance for cardiovascular pathophysiology.Am J Physiol Regul Integr Comp Physiol. 2004; 287: R1014-R1030Crossref PubMed Scopus (679) Google Scholar the reduced NO bioavailability in the cirrhotic liver would be further aggravated by NO scavenging due to its binding to superoxide, which is increased in several liver disorders.10Gracia-Sancho J. Laviña B. Rodriguez-Vilarrupla A. et al.Oxidative stress reduces nitric oxide biodisponibility and may contribute to endothelial dysfunction of cirrhotic livers.J Hepatol. 2006; 44: S75Abstract Full Text PDF Google Scholar, 12Kono H. Rusyn I. Yin M. et al.NADPH oxidase–derived free radicals are key oxidants in alcohol-induced liver disease.J Clin Invest. 2000; 106: 867-872Crossref PubMed Scopus (435) Google Scholar, 13Bataller R. Schwabe R.F. Choi Y.H. et al.NADPH oxidase signal transduces angiotensin II in hepatic stellate cells and is critical in hepatic fibrosis.J Clin Invest. 2003; 112: 1383-1394Crossref PubMed Scopus (508) Google Scholar, 14Carmiel-Haggai M. Cederbaum A.I. Nieto N. A high-fat diet leads to the progression of non-alcoholic fatty liver disease in obese rats.FASEB J. 2005; 19: 136-138Crossref PubMed Scopus (274) Google Scholar, 15Harada H. Hines I.N. Flores S. et al.Role of NADPH oxidase–derived superoxide in reduced size liver ischemia and reperfusion injury.Arch Biochem Biophys. 2004; 423: 103-108Crossref PubMed Scopus (56) Google Scholar It could therefore be possible to increase NO bioavailability and ameliorate hepatic vascular tone by reducing hepatic superoxide. Supporting this concept, it has been shown that the acute administration of high doses of the potent antioxidant vitamin C is able to improve hepatic endothelial dysfunction in patients with cirrhosis.11Hernandez-Guerra M. Garcia-Pagan J.C. Turnes J. et al.Ascorbic acid improves the intrahepatic endothelial dysfunction of patients with cirrhosis and portal hypertension.Hepatology. 2006; 43: 485-491Crossref PubMed Scopus (99) Google Scholar NADPH oxidase is an important source of superoxide that is well characterized as a key enzyme determining endothelial dysfunction in cardiovascular diseases.7Cai H. Harrison D.G. Endothelial dysfunction in cardiovascular diseases: the role of oxidant stress.Circ Res. 2000; 87: 840-844Crossref PubMed Scopus (3272) Google Scholar, 8Hink U. Li H. Mollnau H. et al.Mechanisms underlying endothelial dysfunction in diabetes mellitus.Circ Res. 2001; 88: E14-E22Crossref PubMed Google Scholar, 9Li J.M. Shah A.M. Endothelial cell superoxide generation: regulation and relevance for cardiovascular pathophysiology.Am J Physiol Regul Integr Comp Physiol. 2004; 287: R1014-R1030Crossref PubMed Scopus (679) Google Scholar In these conditions, activation of the enzymatic NADPH oxidase complex generates large amounts of superoxide radical that reacts with NO, reducing its bioavailability.26Cave A.C. Brewer A.C. Narayanapanicker A. et al.NADPH oxidases in cardiovascular health and disease.Antioxid Redox Signal. 2006; 8: 691-728Crossref PubMed Scopus (529) Google Scholar Accordingly, the aims of the current study were to study the molecular and biochemical state of NADPH oxidase in cirrhotic livers and to investigate its possible role modulating hepatic vascular tone in cirrhosis. NADPH oxidase activity was evaluated by the lucigenin-enhanced chemiluminescence assay. Contrary to our hypothesis, NADPH oxidase activity was significantly lower in cirrhotic than in controls rat livers. This result is quite robust, as it was confirmed in 3 independent experiments (with a total of 9 livers per each group). In accordance with this finding, we observed that effective inhibition of NADPH oxidase with apocynin did not cause any significant effect on hepatic or systemic hemodynamics in rats with established cirrhosis. Thus, it appears that in advanced liver disease the NADPH oxidase complex plays no role promoting or aggravating the abnormally elevated hepatic vascular tone and impaired vasorelaxation characteristic of cirrhosis. However, in early stages of the disease, where inflammation processes are more relevant, NADPH oxidase might be increased. In fact, it has been shown that hepatic stellate cell activation by various humoral stimuli, as it happens at the initial stage of fibrogenesis, is NADPH oxidase–mediated and that mice lacking NADPH oxidase are protected from liver fibrosis development.13Bataller R. Schwabe R.F. Choi Y.H. et al.NADPH oxidase signal transduces angiotensin II in hepatic stellate cells and is critical in hepatic fibrosis.J Clin Invest. 2003; 112: 1383-1394Crossref PubMed Scopus (508) Google Scholar, 27Adachi T. Togashi H. Suzuki A. et al.NAD(P)H oxidase plays a crucial role in PDGF-induced proliferation of hepatic stellate cells.Hepatology. 2005; 41: 1272-1281Crossref PubMed Scopus (156) Google Scholar Additionally, in nonalcoholic fatty liver disease, hepatic NADPH oxidase activity and NADPH oxidase expression have been shown to be increased and implicated in the fibrotic process.12Kono H. Rusyn I. Yin M. et al.NADPH oxidase–derived free radicals are key oxidants in alcohol-induced liver disease.J Clin Invest. 2000; 106: 867-872Crossref PubMed Scopus (435) Google Scholar, 27Adachi T. Togashi H. Suzuki A. et al.NAD(P)H oxidase plays a crucial role in PDGF-induced proliferation of hepatic stellate cells.Hepatology. 2005; 41: 1272-1281Crossref PubMed Scopus (156) Google Scholar, 28Gujral J.S. Hinson J.A. Farhood A. et al.NADPH oxidase-derived oxidant stress is critical for neutrophil cytotoxicity during endotoxemia.Am J Physiol Gastrointest Liver Physiol. 2004; 287: G243-G252Crossref PubMed Scopus (104) Google Scholar, 29Colmenero J. Bataller R. Sancho-Bru P. et al.Hepatic expression of candidate genes in patients with alcoholic hepatitis: correlation with disease severity.Gastroenterology. 2007; 132: 687-697Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar Similarly, our group described that development of portal hypertension in the partial portal vein ligation model is associated with increased NADPH oxidase activity in the mesenteric territory and that early NADPH oxidase inhibition prevents splanchnic angiogenesis in this model.30Angermayr B. Fernandez M. Mejias M. et al.NAD(P)H oxidase modulates angiogenesis and the development of portosystemic collaterals and splanchnic hyperaemia in portal hypertensive rats.Gut. 2007; 56: 560-564Crossref PubMed Scopus (51) Google Scholar However, the results of the current study clearly show that NADPH oxidase cannot be considered a player in the intrahepatic hemodynamic abnormalities observed in well established cirrhosis. To understand why cirrhotic livers exhibited low NADPH oxidase activity, we studied the hepatic gene and protein expression of this enzymatic complex. Protein expression of the NADPH oxidase subunits NOX2 and NOX4 was significantly down-regulated in cirrhotic rat livers in comparison with controls, and we could not detect expression of p22phox. Reduced protein expression of NOX4 in cirrhotic livers is remarkable because its expression has been well described in vascular endothelium.26Cave A.C. Brewer A.C. Narayanapanicker A. et al.NADPH oxidases in cardiovascular health and disease.Antioxid Redox Signal. 2006; 8: 691-728Crossref PubMed Scopus (529) Google Scholar, 31Vignais P.V. The superoxide-generating NADPH oxidase: structural aspects and activation mechanism.Cell Mol Life Sci. 2002; 59: 1428-1459Crossref PubMed Scopus (654) Google Scholar, 32Cai H. Griendling K.K. Harrison D.G. The vascular NAD(P)H oxidases as therapeutic targets in cardiovascular diseases.Trends Pharmacol Sci. 2003; 24: 471-478Abstract Full Text Full Text PDF PubMed Scopus (621) Google Scholar, 33Brandes R.P. Kreuzer J. Vascular NADPH oxidases: molecular mechanisms of activation.Cardiovasc Res. 2005; 65: 16-27Crossref PubMed Scopus (333) Google Scholar, 34Van Buul J.D. Fernandez-Borja M. Anthony E.C. et al.Expression and localization of NOX2 and NOX4 in primary human endothelial cells.Antioxid Redox Signal. 2005; 7: 308-317Crossref PubMed Scopus (281) Google Scholar In fact, NOX4 down-regulation has been shown to markedly reduce superoxide production in endothelial cells both in vivo and in vitro, suggesting that NOX4 is a major catalytic component of endothelial NADPH oxidase.35Ago T. Kitazono T. Ooboshi H. et al.Nox4 as the major catalytic component of an endothelial NAD(P)H oxidase.Circulation. 2004; 109: 227-233Crossref PubMed Scopus (443) Google Scholar The reduced protein expression of all the subunits evaluated is in accordance and explains our finding of reduced NADPH oxidase activity in cirrhotic tissue, as well as the lack of effect of NADPH oxidase inhibition. For some of the studies, subunits (NOX4 and NOX1) reduced/absent protein expression was likely due to reduced/absent mRNA expression. However, and unexpectedly, p47phox, p67phox, p22phox, and NOX2 mRNA expression was increased in cirrhotic livers in comparison with controls, despite low NOX2 and no p22phox protein expression, suggesting that in cirrhotic livers mRNA transcription of these subunits could be deregulated. Measurement of NADPH oxidase activity or expression on the whole liver might not be representative of what happens in the hepatic vascular system. Unfortunately, and contrary to what happens with arteries or veins of different vascular territories, because of the architectural nature of the liver it is not feasible to isolate the intrahepatic vascular bed (where most of the hepatic resistance to portal blood flow is generated) to be able to selectively characterize molecular and biochemically the NADPH oxidase system. However, the finding of NOX4 down expression (the main NADPH oxidase subunit expressed in endothelial cells and not in Kupffer cells or macrophages) is the best molecular approach to characterize the NADPH oxidase in the intrahepatic vascular system argues against a role for NADPH oxidase in this setting. Nevertheless, the most convincing evidence that NADPH oxidase does not play a role in modulating the intrahepatic vascular tone in cirrhosis is the physiological studies showing lack of effect of NADPH oxidase inhibition with apocynin. It is always risky to extrapolate data between experimental models; it is even riskier to extrapolate data from experimental models to humans. Thus, to check if there is a rationale to think that our observations in CCl4 cirrhotic rats are also feasible in human cirrhosis, NADPH oxidase enzymatic activity and protein expression were evaluated in liver samples from cirrhotic and control patients. NADPH oxidase enzymatic activity and NOX2 protein expression were not increased in human cirrhotic livers; furthermore, NOX4 protein expression was significantly reduced, thus supporting the lack of role for NADPH oxidase in established cirrhosis. In conclusion, the results of the current study clearly show that NADPH oxidase activity is decreased in cirrhotic livers and therefore cannot explain increased hepatic superoxide radical, endothelial dysfunction, and increased vascular tone in cirrhotic livers. Furthermore, this study opens the rationale to challenge and clarify the role of this enzyme in other liver diseases. The authors are indebted to Héctor García-Calderó and Montse Monclús for technical assistance.
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