Nitroflurbiprofen, a Nitric Oxide-Releasing Cyclooxygenase Inhibitor, Improves Cirrhotic Portal Hypertension in Rats
2006; Elsevier BV; Volume: 132; Issue: 2 Linguagem: Inglês
10.1053/j.gastro.2006.12.041
ISSN1528-0012
AutoresWim Laleman, Lien Van Landeghem, Ingrid Van der Elst, Marcel Zeegers, Johan Fevery, Frederik Nevens,
Tópico(s)Eicosanoids and Hypertension Pharmacology
ResumoBackground & Aims: We studied whether administration of nitroflurbiprofen (HCT-1026), a cyclooxygenase inhibitor with nitric oxide (NO)-donating properties, modulates the increased intrahepatic vascular tone in portal hypertensive cirrhotic rats. Methods: In vivo hemodynamic measurements (n = 8/condition) and evaluation of the increased intrahepatic resistance by in situ perfusion (n = 5/condition) were performed in rats with thioacetamide-induced cirrhosis that received either nitroflurbiprofen (45 mg/kg), flurbiprofen (30 mg/kg, equimolar concentration to nitroflurbiprofen), or vehicle by intraperitoneal injection 24 hours and 1 hour prior to the measurements. Additionally, we evaluated the effect of acute administration of both drugs (250 μmol/L) on the intrahepatic vascular tone in the in situ perfused cirrhotic rat liver (endothelial dysfunction and hyperresponsiveness to methoxamine) and on hepatic stellate cell contraction in vitro. Typical systemic adverse effects of nonsteroidal anti-inflammatory drugs, such as gastrointestinal ulceration, renal insufficiency, and hepatotoxicity, were actively explored. Results: In vivo, nitroflurbiprofen and flurbiprofen equally decreased portal pressure (8 ± 0.8 and 8.4 ± 0.1 mm Hg, respectively, vs 11.8 ± 0.6 mm Hg) and reduced the total intrahepatic vascular resistance. Systemic hypotension was not aggravated in the different treatment groups (P = .291). In the perfused cirrhotic liver, both drugs improved endothelial dysfunction and hyperresponsiveness. This was associated with a decreased hepatic thromboxane A2-production and an increased intrahepatic nitrate/nitrite level. In vitro, nitroflurbiprofen, more than flurbiprofen, decreased hepatic stellate cells contraction. Flurbiprofen-treated rats showed severe gastrointestinal ulcerations (bleeding in 3/8 rats) and nefrotoxicity, which was not observed in nitroflurbiprofen-treated cirrhotic rats. Conclusions: Treatment with nitroflurbiprofen, an NO-releasing cyclooxygenase inhibitor, improves portal hypertension without major adverse effects in thioacetamide-induced cirrhotic rats by attenuating intrahepatic vascular resistance, endothelial dysfunction, and hepatic hyperreactivity to vasoconstrictors. Background & Aims: We studied whether administration of nitroflurbiprofen (HCT-1026), a cyclooxygenase inhibitor with nitric oxide (NO)-donating properties, modulates the increased intrahepatic vascular tone in portal hypertensive cirrhotic rats. Methods: In vivo hemodynamic measurements (n = 8/condition) and evaluation of the increased intrahepatic resistance by in situ perfusion (n = 5/condition) were performed in rats with thioacetamide-induced cirrhosis that received either nitroflurbiprofen (45 mg/kg), flurbiprofen (30 mg/kg, equimolar concentration to nitroflurbiprofen), or vehicle by intraperitoneal injection 24 hours and 1 hour prior to the measurements. Additionally, we evaluated the effect of acute administration of both drugs (250 μmol/L) on the intrahepatic vascular tone in the in situ perfused cirrhotic rat liver (endothelial dysfunction and hyperresponsiveness to methoxamine) and on hepatic stellate cell contraction in vitro. Typical systemic adverse effects of nonsteroidal anti-inflammatory drugs, such as gastrointestinal ulceration, renal insufficiency, and hepatotoxicity, were actively explored. Results: In vivo, nitroflurbiprofen and flurbiprofen equally decreased portal pressure (8 ± 0.8 and 8.4 ± 0.1 mm Hg, respectively, vs 11.8 ± 0.6 mm Hg) and reduced the total intrahepatic vascular resistance. Systemic hypotension was not aggravated in the different treatment groups (P = .291). In the perfused cirrhotic liver, both drugs improved endothelial dysfunction and hyperresponsiveness. This was associated with a decreased hepatic thromboxane A2-production and an increased intrahepatic nitrate/nitrite level. In vitro, nitroflurbiprofen, more than flurbiprofen, decreased hepatic stellate cells contraction. Flurbiprofen-treated rats showed severe gastrointestinal ulcerations (bleeding in 3/8 rats) and nefrotoxicity, which was not observed in nitroflurbiprofen-treated cirrhotic rats. Conclusions: Treatment with nitroflurbiprofen, an NO-releasing cyclooxygenase inhibitor, improves portal hypertension without major adverse effects in thioacetamide-induced cirrhotic rats by attenuating intrahepatic vascular resistance, endothelial dysfunction, and hepatic hyperreactivity to vasoconstrictors. Although the knowledge concerning the pathophysiology of portal hypertension has improved tremendously over the past 25 years, the therapeutic armamentarium has not assumed equal proportions.1Laleman W. Van Landeghem L. Wilmer A. Fevery J. Nevens F. Portal hypertension: from pathophysiology to clinical practice.Liver Int. 2005; 25: 1079-1090Crossref PubMed Scopus (120) Google Scholar As a result, nonselective β-blockers, since their introduction more than 20 years ago, have remained the mainstay of pharmacologic therapy for portal hypertension.1Laleman W. Van Landeghem L. Wilmer A. Fevery J. Nevens F. Portal hypertension: from pathophysiology to clinical practice.Liver Int. 2005; 25: 1079-1090Crossref PubMed Scopus (120) Google Scholar, 2Lebrec D. Poynard T. Hillon P. Benhamou J.P. Propanolol for the prevention of recurrent gastrointestinal bleeding in patients with cirrhosis: a controlled study.N Engl J Med. 1981; 305: 1371-1374Crossref PubMed Scopus (419) Google Scholar, 3Pascal J.P. Cales P. Propanolol in the prevention of first upper gastrointestinal tract hemorrhage in patients with cirrhosis of the liver and esophageal varices.N Engl J Med. 1987; 317: 856-861Crossref PubMed Scopus (273) Google Scholar, 4Abraldes J.G. Tarantino I. Turnes J. Garcia-Pagan J.C. Rodes J. Bosch J. Hemodynamic response to pharmacological treatment of portal hypertension and long-term prognosis of cirrhosis.Hepatology. 2003; 37: 902-908Crossref PubMed Scopus (397) Google Scholar Undeniably, these drugs have proven to be of value because they have reduced the risk of variceal (re)bleeding and bleeding-related mortality on a short-term basis. In addition, they also decrease the risk of developing long-term complications, such as ascites, spontaneous bacterial peritonitis, and hepatorenal syndrome, and improve long-term survival.4Abraldes J.G. Tarantino I. Turnes J. Garcia-Pagan J.C. Rodes J. Bosch J. Hemodynamic response to pharmacological treatment of portal hypertension and long-term prognosis of cirrhosis.Hepatology. 2003; 37: 902-908Crossref PubMed Scopus (397) Google Scholar, 5Groszmann R.J. Bosch J. Grace N.D. Conn H.O. Garcia-Tsao G. Navasa M. Alberts J. Rodes J. Fischer R. Bermann M. Hemodynamic events in a prospective randomized trial of propranolol versus placebo in the prevention of a first variceal hemorrhage.Gastroenterology. 1990; 90: 1401-1407Google Scholar, 6Feu F. Garcia-Pagan J.C. Bosch J. Luca A. Teres J. Escorsell A. Rodes J. Relation between portal pressure response to pharmacotherapy and risk of recurrent variceal haemorrhage in patients with cirrhosis.Lancet. 1995; 346: 1056-1059Abstract Full Text PDF PubMed Scopus (503) Google Scholar, 7Bosch J. Garcia-Pagan J.C. Prevention of variceal rebleeding.Lancet. 2003; 361: 952-954Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar Nonetheless, a sustained decrease in portal pressure is only achieved in less than half of the treated patients, urging the need for potential new or additive drugs.1Laleman W. Van Landeghem L. Wilmer A. Fevery J. Nevens F. Portal hypertension: from pathophysiology to clinical practice.Liver Int. 2005; 25: 1079-1090Crossref PubMed Scopus (120) Google Scholar, 6Feu F. Garcia-Pagan J.C. Bosch J. Luca A. Teres J. Escorsell A. Rodes J. Relation between portal pressure response to pharmacotherapy and risk of recurrent variceal haemorrhage in patients with cirrhosis.Lancet. 1995; 346: 1056-1059Abstract Full Text PDF PubMed Scopus (503) Google Scholar, 8Villeneuve J.P. Pomier-Layrargues G. Infante-Rivard C. Willems B. Huet P.M. Marleau D. Viallet A. Propranolol for the prevention of recurrent variceal hemorrhage: a controlled trial.Hepatology. 1986; 6: 1239-1243Crossref PubMed Scopus (123) Google Scholar Recent advances in the knowledge of the pathophysiology of cirrhotic portal hypertension have directed future therapy toward the increased intrahepatic vascular resistance, which in part is determined by an increased hepatic vascular tone.9Bhathal P.S. Grossman H.J. Reduction of the increased intrahepatic vascular resistance of the isolated perfused cirrhotic rat liver by vasodilators.J Hepatol. 1985; 1: 325-337Abstract Full Text PDF PubMed Scopus (295) Google Scholar This increased vascular tone is mainly driven by activated hepatic stellate cells (HSCs) and results from an imbalance between vasodilator and vasoconstrictor forces.1Laleman W. Van Landeghem L. Wilmer A. Fevery J. Nevens F. Portal hypertension: from pathophysiology to clinical practice.Liver Int. 2005; 25: 1079-1090Crossref PubMed Scopus (120) Google Scholar, 10Rockey D.C. The cellular pathogenesis of portal hypertension: stellate cell contractility, endothelin and nitric oxide.Hepatology. 1997; 25: 2-5Crossref PubMed Scopus (306) Google Scholar More specifically, there is an increased intrahepatic production of vasoconstrictors, including thromboxane A2 (TXA2), a cyclooxygenase (COX)-derived prostanoid, which has recently been suggested to play a major role in the intrahepatic endothelial dysfunction and the aggravated response to vasoconstrictors (“hyperresponsiveness”) in the cirrhotic liver.10Rockey D.C. The cellular pathogenesis of portal hypertension: stellate cell contractility, endothelin and nitric oxide.Hepatology. 1997; 25: 2-5Crossref PubMed Scopus (306) Google Scholar, 11Graupera M. Garcia-Pagan J.C. Abraldes J.G. Peralta C. Bragulat M. Corominola H. Bosch J. Rodes J. Cyclooxygenase-derived products modulate the increased intrahepatic resistance of cirrhotic rat livers.Hepatology. 2003; 37: 172-181Crossref PubMed Scopus (117) Google Scholar, 12Graupera M. Garcia-Pagan J.C. Pares M. Abraldes J.G. Rosello J. Bosch J. Rodes J. Cyclooxygenase-1 inhibition corrects endothelial dysfunction in cirrhotic rat livers.J Hepatol. 2003; 39: 515-521Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar Additionally, there is decreased availability of vasodilators, of which nitric oxide (NO) is the most renowned.13Wiest R. Groszmann R.J. The paradox of nitric oxide in cirrhosis and portal hypertension: too much, not enough.Hepatology. 2002; 35: 478-491Crossref PubMed Scopus (370) Google Scholar, 14Gupta T.K. Toruner M. Chung M.K. Groszmann R.J. Endothelial dysfunction and decreased production of nitric oxide in the intrahepatic microcirculation of cirrhotic rats.Hepatology. 1998; 28: 926-931Crossref PubMed Scopus (307) Google Scholar, 15Van De Casteele M. Van Pelt J.F. Nevens F. Fevery J. Reichen J. 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, 16Van de Casteele M. Omasta A. Janssens S. Roskams T. Desmet V. Nevens F. Fevery J. In vivo gene transfer of endothelial nitric oxide synthase decreases portal pressure in anaesthetised carbon tetrachloride cirrhotic rats.Gut. 2002; 51: 440-445Crossref PubMed Scopus (79) Google Scholar, 17Laleman W. Omasta A. Van de Casteele M. Zeegers M. Vander Elst I. Van Landeghem L. Severi T. van Pelt J. Roskams T. Fevery J. Nevens F. A role for asymmetric dimethylarginine in the pathophysiology of portal hypertension in rats with biliary cirrhosis.Hepatology. 2005; 42: 1382-1390Crossref PubMed Scopus (66) Google Scholar A possible pharmacologic strategy in the treatment of portal hypertension would therefore be to abrogate the generation of TXA2 in combination with a replenishment of the decreased hepatic NO bioavailability. The vasodilatory effect of this drug, however, should remain limited to the hepatic microcirculation to avoid further worsening of the associated splanchnic and systemic vasodilatory syndrome, in which increased biosynthesis of NO and the vasodilator COX metabolite prostacyclin PGI2 have been implicated.18Fernandez M. Garcia-Pagan J.C. Casadevall M. Mourelle M.I. Pique J.M. Bosch J. Rodes J. Acute and chronic cyclooxygenase blockade in portal-hypertensive rats: influence on nitric oxide biosynthesis.Gastroenterology. 1996; 110: 1529-1535Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 19Hou M.C. Cahill P.A. Zhang S. Wang Y.N. Hendrickson R.J. Redmond E.M. Sitzmann J.V. Enhanced cyclooxygenase-1 expression within the superior mesenteric artery of portal hypertensive rats: role in the hyperdynamic circulation.Hepatology. 1998; 27: 20-27Crossref PubMed Scopus (47) Google Scholar This everlasting conflicting paradox of “too much, not enough” is currently the major impediment to the use of nitrovasodilators such as isosorbide-5-mononitrate.1Laleman W. Van Landeghem L. Wilmer A. Fevery J. Nevens F. Portal hypertension: from pathophysiology to clinical practice.Liver Int. 2005; 25: 1079-1090Crossref PubMed Scopus (120) Google Scholar, 13Wiest R. Groszmann R.J. The paradox of nitric oxide in cirrhosis and portal hypertension: too much, not enough.Hepatology. 2002; 35: 478-491Crossref PubMed Scopus (370) Google Scholar In the present study, we examined the hemodynamic effect and safety profile of nitroflurbiprofen (HCT-1026), a COX inhibitor with selective NO-donating properties, in rats with established cirrhotic portal hypertension and associated hyperdynamic circulation. Male Wistar rats (Animal House, University of Leuven, Leuven, Belgium), weighing 200–250 g, were used. Experiments and procedures were performed in accordance with local animal care guidelines. All studies were performed in cirrhotic animals (n = 74 in total) that underwent chronic exposure to thioacetamide (TAA), given in drinking water for 18 weeks, as described.17Laleman W. Omasta A. Van de Casteele M. Zeegers M. Vander Elst I. Van Landeghem L. Severi T. van Pelt J. Roskams T. Fevery J. Nevens F. A role for asymmetric dimethylarginine in the pathophysiology of portal hypertension in rats with biliary cirrhosis.Hepatology. 2005; 42: 1382-1390Crossref PubMed Scopus (66) Google Scholar, 20Laleman W. Vander Elst I. Zeegers M. Servaes R. Libbrecht L. Roskams T. Fevery J. Nevens F. A stable model of cirrhotic portal hypertension in the rat: thioacetamide revisited.Eur J Clin Invest. 2006; 36: 242-249Crossref PubMed Scopus (51) Google Scholar Normal rats were used as internal controls in the perfusion experiments (n = 15) and for the isolation of HSC (n = 3). After 18 weeks of TAA administration, cirrhotic rats were equally (n = 8/group) and randomly allocated to one of the following groups: intraperitoneal injection, 24 hours and 1 hour prior to the measurements, with nitroflurbiprofen (45 mg/kg) (HCT-1026; Nicox, Sophia Antipolis Cedex, France), flurbiprofen (30 mg/kg, equimolar concentration to nitroflurbiprofen) (Sigma Chemical Co, St. Louis, MO), or vehicle (250 μL dimethylsulfoxide [DMSO]:250 μL isotonic saline). The used dose was based on a dose-finding study (n = 5 per condition). We started with concentrations of nitroflurbiprofen (15 mg/kg) and flurbiprofen (7.5 mg/kg, equimolar concentration to nitroflurbiprofen flurbiprofen), increased the dose to nitroflurbiprofen 22.5 mg/kg and flurbiprofen 15 mg/kg, and, finally, nitroflurbiprofen 45 mg/kg and flurbiprofen 30 mg/kg. The last dose regimen had the most profound hemodynamic effects. Therefore, the currently described experiments were performed with the latter doses of nitroflurbiprofen and flurbiprofen, respectively. Afterwards, rats were instrumented for in vivo hemodynamic measurements. Measurements were carried out under pentobarbital anesthesia to allow cannulation of the carotid artery and portal vein, as described.16Van de Casteele M. Omasta A. Janssens S. Roskams T. Desmet V. Nevens F. Fevery J. In vivo gene transfer of endothelial nitric oxide synthase decreases portal pressure in anaesthetised carbon tetrachloride cirrhotic rats.Gut. 2002; 51: 440-445Crossref PubMed Scopus (79) Google Scholar, 17Laleman W. Omasta A. Van de Casteele M. Zeegers M. Vander Elst I. Van Landeghem L. Severi T. van Pelt J. Roskams T. Fevery J. Nevens F. A role for asymmetric dimethylarginine in the pathophysiology of portal hypertension in rats with biliary cirrhosis.Hepatology. 2005; 42: 1382-1390Crossref PubMed Scopus (66) Google Scholar, 20Laleman W. Vander Elst I. Zeegers M. Servaes R. Libbrecht L. Roskams T. Fevery J. Nevens F. A stable model of cirrhotic portal hypertension in the rat: thioacetamide revisited.Eur J Clin Invest. 2006; 36: 242-249Crossref PubMed Scopus (51) Google Scholar Flow in the superior mesenteric artery (mesenteric blood flow; MBF) was determined by use of a 1-mm nonconstrictive perivascular flow probe (1RB, Transonic, Ithaca, NY) connected to a T-206 flowmeter, as described.17Laleman W. Omasta A. Van de Casteele M. Zeegers M. Vander Elst I. Van Landeghem L. Severi T. van Pelt J. Roskams T. Fevery J. Nevens F. A role for asymmetric dimethylarginine in the pathophysiology of portal hypertension in rats with biliary cirrhosis.Hepatology. 2005; 42: 1382-1390Crossref PubMed Scopus (66) Google Scholar, 18Fernandez M. Garcia-Pagan J.C. Casadevall M. Mourelle M.I. Pique J.M. Bosch J. Rodes J. Acute and chronic cyclooxygenase blockade in portal-hypertensive rats: influence on nitric oxide biosynthesis.Gastroenterology. 1996; 110: 1529-1535Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 20Laleman W. Vander Elst I. Zeegers M. Servaes R. Libbrecht L. Roskams T. Fevery J. Nevens F. A stable model of cirrhotic portal hypertension in the rat: thioacetamide revisited.Eur J Clin Invest. 2006; 36: 242-249Crossref PubMed Scopus (51) Google Scholar For the in situ liver perfusion studies, a flow-controlled system was used, as described.11Graupera M. Garcia-Pagan J.C. Abraldes J.G. Peralta C. Bragulat M. Corominola H. Bosch J. Rodes J. Cyclooxygenase-derived products modulate the increased intrahepatic resistance of cirrhotic rat livers.Hepatology. 2003; 37: 172-181Crossref PubMed Scopus (117) Google Scholar, 12Graupera M. Garcia-Pagan J.C. Pares M. Abraldes J.G. Rosello J. Bosch J. Rodes J. Cyclooxygenase-1 inhibition corrects endothelial dysfunction in cirrhotic rat livers.J Hepatol. 2003; 39: 515-521Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 14Gupta T.K. Toruner M. Chung M.K. Groszmann R.J. Endothelial dysfunction and decreased production of nitric oxide in the intrahepatic microcirculation of cirrhotic rats.Hepatology. 1998; 28: 926-931Crossref PubMed Scopus (307) Google Scholar, 17Laleman W. Omasta A. Van de Casteele M. Zeegers M. Vander Elst I. Van Landeghem L. Severi T. van Pelt J. Roskams T. Fevery J. Nevens F. A role for asymmetric dimethylarginine in the pathophysiology of portal hypertension in rats with biliary cirrhosis.Hepatology. 2005; 42: 1382-1390Crossref PubMed Scopus (66) Google Scholar, 20Laleman W. Vander Elst I. Zeegers M. Servaes R. Libbrecht L. Roskams T. Fevery J. Nevens F. A stable model of cirrhotic portal hypertension in the rat: thioacetamide revisited.Eur J Clin Invest. 2006; 36: 242-249Crossref PubMed Scopus (51) Google Scholar, 21Cales P. Oberti F. Veal N. Fort J. Kaassis M. Moal F. Aube C. Vuillenin E. Pilette C. Rifflet H. Trouve R. Splenorenal shunt blood flow by transit time ultrasound as an index of collateral circulation in portal hypertensive rats.Hepatology. 1998; 28: 1269-1274Crossref PubMed Scopus (12) Google Scholar After laparotomy, a ligature was passed around the suprarenal portion of the inferior vena cava, which was injected with 1400 U heparin/kg body weight. The portal vein was cannulated with a 14-gauge catheter (Becton-Dickinson, Erembodegem, Belgium) and the liver was perfused with Krebs solution pumped (Masterflex, Vernon Hills, IL) from the reservoir into an overflow chamber via a PALL Ultipor filter (Pall, East Hills, NY) and oxygenated via a silastic tubing oxygenator (Sophysa, Nijvel, Belgium) at 37°C ± 0.5°C. The abdominal aorta and inferior vena cava were cut below the ligature, allowing the exsanguination of the animal and the escape of the perfusate. Following thoracotomy, the right atrium was cannulated (PE-240; Portex, Kent, United Kingdom) advancing the catheter through the inferior vena cava up to the outlet of the hepatic vein. When the perfused effluent was clear, the system was closed and recirculation obtained with a volume of 150 mL at a constant flow of 35 mL/min. An ultrasonic flow probe (T201, Transonic Systems) and a pressure gauge were placed in line, immediately upstream of the inlet cannula, to monitor continuously the portal flow and perfusion pressure. Criteria of liver viability included gross appearance of the liver, stable perfusion pressure (starting value ± 1 mm Hg), and a stable buffer pH (7.4 ± 0.1) during an initial 30-minute stabilization period. The effect of intraperitoneal injection, 24 hours and 1 hour prior to measurement, of nitroflurbiprofen (45 mg/kg), flurbiprofen (30 mg/kg), or vehicle on the total intrahepatic vascular resistance was evaluated in 15 additional rats (n = 5/group) by performing flow-pressure curves (increase with 5 mL/min every 5 minutes) (n = 5 for each condition). To test the direct effect of these drugs on the increased intrahepatic vascular tone, nitroflurbiprofen (250 μmol/L), flurbiprofen (250 μmol/L), or vehicle (DMSO:isotonic saline) were added to the perfusate of an additional group of treatment-naive perfused cirrhotic rats (n = 30 in total) after the stabilization period. In one set of experiments, L-NAME, a nitric oxide synthase (NOS)-inhibitor, was preincubated together with flurbiprofen, a COX-inhibitor. Thereafter, the preparation was allowed to stabilize for 15 minutes. The increased intrahepatic vascular tone was assessed through the evaluation of endothelial function (n = 5 per condition) and responsiveness to vasoconstrictors (n = 5 per condition). More specifically, endothelial function was evaluated by performing concentration-effect curves to cumulative doses of acetylcholine (10−7 to 10−5 mol/L, one log increase every 1.5 minutes) (Sigma) after a 5-minute precontraction of the liver with the α1-adrenergic agonist methoxamine 10−4 mol/L (Sigma),12Graupera M. Garcia-Pagan J.C. Pares M. Abraldes J.G. Rosello J. Bosch J. Rodes J. Cyclooxygenase-1 inhibition corrects endothelial dysfunction in cirrhotic rat livers.J Hepatol. 2003; 39: 515-521Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 14Gupta T.K. Toruner M. Chung M.K. Groszmann R.J. Endothelial dysfunction and decreased production of nitric oxide in the intrahepatic microcirculation of cirrhotic rats.Hepatology. 1998; 28: 926-931Crossref PubMed Scopus (307) Google Scholar, 17Laleman W. Omasta A. Van de Casteele M. Zeegers M. Vander Elst I. Van Landeghem L. Severi T. van Pelt J. Roskams T. Fevery J. Nevens F. A role for asymmetric dimethylarginine in the pathophysiology of portal hypertension in rats with biliary cirrhosis.Hepatology. 2005; 42: 1382-1390Crossref PubMed Scopus (66) Google Scholar, 20Laleman W. Vander Elst I. Zeegers M. Servaes R. Libbrecht L. Roskams T. Fevery J. Nevens F. A stable model of cirrhotic portal hypertension in the rat: thioacetamide revisited.Eur J Clin Invest. 2006; 36: 242-249Crossref PubMed Scopus (51) Google Scholar whereas responsiveness to vasoconstrictors was tested by performing dose-response curves to cumulative doses of methoxamine (10−6 to 10−4 mol/L, one log increase every 5 minutes).11Graupera M. Garcia-Pagan J.C. Abraldes J.G. Peralta C. Bragulat M. Corominola H. Bosch J. Rodes J. Cyclooxygenase-derived products modulate the increased intrahepatic resistance of cirrhotic rat livers.Hepatology. 2003; 37: 172-181Crossref PubMed Scopus (117) Google Scholar, 20Laleman W. Vander Elst I. Zeegers M. Servaes R. Libbrecht L. Roskams T. Fevery J. Nevens F. A stable model of cirrhotic portal hypertension in the rat: thioacetamide revisited.Eur J Clin Invest. 2006; 36: 242-249Crossref PubMed Scopus (51) Google Scholar In the experiments assessing endothelial function (concentration-effect curves to cumulative doses of acetylcholine), 2-mL samples of the perfusate were taken before and after the experiment for the measurement of thromboxane B (TXB)2 production (Caymann Chem, Ann Arbor, MI), a stable metabolite of TXA2. TXB2 production was expressed as the absolute increment over baseline before methoxamine administration. HSCs were isolated, as described previously.22Cassiman D. Denef C. Desmet V.J. Roskams T. Human and rat hepatic stellate cells express neurotrophins and neurotrophin receptors.Hepatology. 2001; 33: 148-158Crossref PubMed Scopus (192) Google Scholar In brief, following in situ perfusion of the liver with collagenase type IV (Sigma) and pronase E (Merck, Darmstadt, Germany) solution, the resulting cell suspension was fractionated by density gradient centrifugation using 9% Optiprep (Nycomed, Sweden). The cells were cultured in William’s E medium supplemented with 10% fetal calf serum (FCS), 0.6 IU/mL insulin, 2 mmol/L glutamine, and 1% antibiotic-antimycotic solution (Invitrogen, Merelbeke, Belgium). The medium was renewed every 48–72 hours. Characterization of the rat liver-derived myofibroblast-like cultures, established by culturing enriched HSC fractions on plastic, was performed by staining with anti-α-smooth muscle actin, antidesmin, and synaptophysin, as described.22Cassiman D. Denef C. Desmet V.J. Roskams T. Human and rat hepatic stellate cells express neurotrophins and neurotrophin receptors.Hepatology. 2001; 33: 148-158Crossref PubMed Scopus (192) Google Scholar, 23Cassiman D. Roskams T. Beauty is in the eye of the beholder: emerging concepts and pitfalls in hepatic stellate cell research.J Hepatol. 2002; 37: 527-535Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar The ability of HSCs to contract 3-dimensional collagen matrices was assessed as previously described, with some modifications.24van Bockxmeer F.M. Martin C.E. Constable I.J. Effect of cyclic AMP on cellular contractility and DNA synthesis in chorioretinal fibroblasts maintained in collagen matrices.Exp Cell Res. 1984; 155: 413-421Crossref PubMed Scopus (32) Google Scholar, 25Grinnell F. Fibroblast biology in three-dimensional collagen matrices.Trends Cell Biol. 2003; 13: 264-269Abstract Full Text Full Text PDF PubMed Scopus (661) Google Scholar In brief, hydrated collagen gels were prepared using rat tail tendon collagen I (Becton-Dickinson, Bedford, MA), adjusted to physiologic strength and pH with 1 N NaOH and 10X phosphate-buffered saline (PBS) at 4°C. Afterwards, the collagen solution was mixed with an HSC suspension so that the final solution resulted in a collagen concentration of 1.5 mg/mL and 250,000 cells/mL. A 500-μL aliquot of the collagen solution was then cast into a 24-well tissue culture plate (Falcon, Meylan, France) and covered after 1 hour with culture medium (1 mL/well) to ascertain adequate gelation. Afterwards, the polymerized collagen matrix containing HSCs remained attached to the culture dish for 24 hours leading to mechanical loading (“stressed”), as such mimicking geometric and mechanical relationships that HSCs entertain in vivo.25Grinnell F. Fibroblast biology in three-dimensional collagen matrices.Trends Cell Biol. 2003; 13: 264-269Abstract Full Text Full Text PDF PubMed Scopus (661) Google Scholar After 24 hours, stabilized lattices were washed with 1X PBS, followed by addition of 1 mL serum-free culture medium per well containing 1 μCi 3H2O (Amersham Biosciences, Roosendaal, The Netherlands). To initiate matrix contraction, the mechanically stressed matrices were released by gentle circumferential dislodgement of the lattice (“relaxation”). Cell-mediated contraction was measured by determining the relative partioning of 3H2O between the gel phase and the surrounding medium following 24 hours of contraction, thereby allowing estimation of gel phase volumes. More specifically, the separate tritium activities of medium and gel phase were measured in 10 mL oscillation fluid (Perkin Elmer, Wellesley, MA) using a Beckmann liquid scintillation spectrometer. Control cell-free gels provided estimates for the precontraction volume and were allowed to express relative changes in volume (percentage contraction). The effect of nitroflurbiprofen (25, 50, 100, and 250 μmol/L), flurbiprofen (250 μmol/L), and vehicle (DMSO) on fetal calf serum (FCS)-promoted contraction was evaluated by preincubating these compounds before addition of FCS 10%. To additionally prove that HSCs were sensitive to TXA2-mediated contraction, a subset of collagen matrices were preincubated with 1 and 2.5 μmol/L SQ29,548 (Caymann Chem) before adding 5 μmol/L U44619, a stable TXA2-analogue (Caymann Chem). All data are from experiments using at least 3 sets of 3 collagen lattices containing culture-activated HSCs from 3 different rat HSC isolations (passage 1 to 3). To determine the degree of systemic NO release, nitrate/nitrite (NOx) content, a parameter of NO production, was assayed by a fluorometric method using 2,3-diaminonaphtalene in the serum of the differently treated groups of cirrhotic rats, used in the in vivo hemodynamic study, as described.17Laleman W. Omasta A. Van de Casteele M. Zeegers M. Vander Elst I. Van Landeghem L. Severi T. van Pelt J. Roskams T. Fevery J. Nevens F. A role for asymmetric dimethylarginine in the pathophysiology of portal hypertension in rats with biliary cirrhosis.Hepatology. 2005;
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