Increased Gene and Protein Expression of the Novel eNOS Regulatory Protein NOSTRIN and a Variant in Alcoholic Hepatitis
2006; Elsevier BV; Volume: 132; Issue: 7 Linguagem: Inglês
10.1053/j.gastro.2006.12.035
ISSN1528-0012
AutoresRajeshwar P. Mookerjee, Anja Wiesenthal, Ann Icking, Stephen Hodges, Nathan Davies, Kirstin Schilling, Sambit Sen, Roger Williams, Marco Novelli, Werner Müller‐Esterl, Rajiv Jalan,
Tópico(s)Eicosanoids and Hypertension Pharmacology
ResumoBackground & Aims: Increased intrahepatic resistance in cirrhosis is associated with reduced endothelial NO synthase (eNOS) activity and exacerbated by superimposed inflammation. NOSTRIN induces intracellular translocation of eNOS and reduces NO generation. Our aims were to quantify and compare hepatic expression of eNOS, NOSTRIN, NOSIP, and caveolin-1 in alcoholic cirrhosis with or without superimposed alcoholic hepatitis and in normal livers. Methods: Biopsy specimens from 20 decompensated alcoholic cirrhotic patients with portal hypertension (10 with alcoholic hepatitis) and 6 normal livers were analyzed: real-time polymerase chain reaction for quantification of messenger RNA; Western blotting; and enzyme assays of eNOS in normal and diseased liver were performed. Localization and interaction of eNOS and NOSTRIN in liver was assessed by immunohistochemistry and co-immunoprecipitation. Results: eNOS mRNA was significantly increased and eNOS activity decreased in alcoholic hepatitis patients, despite no differences in eNOS protein expression among the patients. Patients with alcoholic hepatitis had significantly higher hepatic levels of NOSTRIN and caveolin-1 mRNA compared with cirrhosis alone or normal biopsy specimens. A NOSTRIN splice variant, not present in normal tissue, was detected on mRNA and protein levels in all alcoholic patients. Coimmunoprecipitation demonstrated association among NOSTRIN, eNOS, and caveolin-1. Conclusions: An increase in mRNA and protein of NOSTRIN and its shortened variant in alcoholic hepatitis may partly account for the paradox of increased mRNA levels and normal protein expression but decreased enzymatic activity of eNOS in diseased liver. Such intracellular regulators of NO production may be important in the development of increased intrahepatic resistance in alcoholic hepatitis patients. Background & Aims: Increased intrahepatic resistance in cirrhosis is associated with reduced endothelial NO synthase (eNOS) activity and exacerbated by superimposed inflammation. NOSTRIN induces intracellular translocation of eNOS and reduces NO generation. Our aims were to quantify and compare hepatic expression of eNOS, NOSTRIN, NOSIP, and caveolin-1 in alcoholic cirrhosis with or without superimposed alcoholic hepatitis and in normal livers. Methods: Biopsy specimens from 20 decompensated alcoholic cirrhotic patients with portal hypertension (10 with alcoholic hepatitis) and 6 normal livers were analyzed: real-time polymerase chain reaction for quantification of messenger RNA; Western blotting; and enzyme assays of eNOS in normal and diseased liver were performed. Localization and interaction of eNOS and NOSTRIN in liver was assessed by immunohistochemistry and co-immunoprecipitation. Results: eNOS mRNA was significantly increased and eNOS activity decreased in alcoholic hepatitis patients, despite no differences in eNOS protein expression among the patients. Patients with alcoholic hepatitis had significantly higher hepatic levels of NOSTRIN and caveolin-1 mRNA compared with cirrhosis alone or normal biopsy specimens. A NOSTRIN splice variant, not present in normal tissue, was detected on mRNA and protein levels in all alcoholic patients. Coimmunoprecipitation demonstrated association among NOSTRIN, eNOS, and caveolin-1. Conclusions: An increase in mRNA and protein of NOSTRIN and its shortened variant in alcoholic hepatitis may partly account for the paradox of increased mRNA levels and normal protein expression but decreased enzymatic activity of eNOS in diseased liver. Such intracellular regulators of NO production may be important in the development of increased intrahepatic resistance in alcoholic hepatitis patients. Patients with severe alcoholic liver disease and cirrhosis often present with complications of portal hypertension including variceal bleeding, sodium retention, and renal dysfunction. The mechanisms underlying the hemodynamic disturbances including elevated portal pressure seen in these patients remain unclear. There is an increasing body of literature in cirrhotic animals suggesting a reduction in the activity of hepatic endothelial nitric oxide synthase (eNOS),1Gupta 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 (304) Google Scholar, 2Rockey 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 (329) Google Scholar and this is despite apparently normal hepatic eNOS protein expression.3Shah V. Toruner M. Haddad F. Cadelina G. Papapetropoulos A. Choo K. Sessa W.C. Groszmann R.J. 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 (274) Google Scholar The resultant reduction in hepatic nitric oxide (NO) generation contributes to increased intrahepatic resistance and elevated portal pressure. Indeed, a reduction in intrahepatic resistance, and thereby severity of portal hypertension, through the delivery of NO to the liver in model systems, either pharmacologically4Fiorucci S. Antonelli E. Brancaleone V. Sanpaolo L. Orlandi S. Distrutti E. Acuto G. Clerici C. Baldoni M. Del Soldato P. Morelli A. NCX-1000, a nitric oxide-releasing derivative of ursodeoxycholic acid, ameliorates portal hypertension and lowers norepinephrine-induced intrahepatic resistance in the isolated and perfused rat liver.J Hepatol. 2003; 39: 932-939Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar or through gene transfer techniques,5Van 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, 6Morales-Ruiz M. Cejudo-Martn P. Fernandez-Varo G. Tugues S. Ros J. Angeli P. Rivera F. Arroyo V. Rodes J. Sessa W.C. Jimenez W. Transduction of the liver with activated Akt normalizes portal pressure in cirrhotic rats.Gastroenterology. 2003; 125: 522-531Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar highlights the importance of hepatic NO generation in the pathophysiology of portal hypertension.Another important concept is the role of additional inflammation superimposed on cirrhosis in increasing the severity of portal hypertension. This is supported by studies demonstrating a reduction in portal pressure in portal hypertensive animals undergoing interventions directed at mediators of inflammation, such as treatment with thalidomide7Lopez-Talavera J.C. Cadelina G. Olchowski J. Merrill W. Groszmann R.J. Thalidomide inhibits tumor necrosis factor α, decreases nitric oxide synthesis, and ameliorates the hyperdynamic circulatory syndrome in portal-hypertensive rats.Hepatology. 1996; 23: 1616-1621PubMed Google Scholar or tumor necrosis factor (TNF)-α-directed monoclonal antibodies.8Lopez-Talavera J.C. Merrill W.W. Groszmann R.J. Tumor necrosis factor α: a major contributor to the hyperdynamic circulation in prehepatic portal-hypertensive rats.Gastroenterology. 1995; 108: 761-767Abstract Full Text PDF PubMed Scopus (158) Google Scholar In keeping with these observations, we found a significant, acute reduction in portal pressure in patients with cirrhosis who had superimposed alcoholic hepatitis (AH), following administration of the anti-TNF-α antibody infliximab.9Mookerjee R.P. Sen S. Davies N.A. Hodges S. Williams R. Jalan R. TNF is an important mediator of portal and systemic haemodynamic derangements in alcoholic hepatitis.Gut. 2003; 52: 1182-1187Crossref PubMed Scopus (126) Google Scholar In addition, the possible removal of mediators of inflammation with extracorporeal albumin dialysis resulted in acute reduction in the severity of portal hypertension in patients with severe alcoholic hepatitis.10Sen S. Mookerjee R.P. Cheshire L.M. Davies N.A. Williams R. Jalan R. Albumin dialysis reduces portal pressure acutely in patients with severe alcoholic hepatitis.J Hepatol. 2005; 43: 142-148Abstract Full Text Full Text PDF PubMed Scopus (82) Google ScholarStudies in relation to decreased hepatic eNOS activity have, to date, focused on inhibitors of eNOS activity such as caveolin-13Shah V. Toruner M. Haddad F. Cadelina G. Papapetropoulos A. Choo K. Sessa W.C. Groszmann R.J. 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 (274) Google Scholar, 11Ashburn J.H. Baveja R. Kresge N. Korneszczuk K. Keller S. Karaa A. Yokoyama Y. Zhang J.X. Huynh T. Clemens M.G. Remote trauma sensitizes hepatic microcirculation to endothelin via caveolin inhibition of eNOS activity.Shock. 2004; 22: 120-130Crossref PubMed Scopus (23) Google Scholar and asymmetric dimethylarginine12Vallance P. Leone A. Calver A. Collier J. Moncada S. Accumulation of an endogenous inhibitor of nitric oxide synthesis in chronic renal failure.Lancet. 1992; 339: 572-575Abstract PubMed Scopus (1940) Google Scholar or on processes affecting the posttranslational modification of eNOS.6Morales-Ruiz M. Cejudo-Martn P. Fernandez-Varo G. Tugues S. Ros J. Angeli P. Rivera F. Arroyo V. Rodes J. Sessa W.C. Jimenez W. Transduction of the liver with activated Akt normalizes portal pressure in cirrhotic rats.Gastroenterology. 2003; 125: 522-531Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar Recently, a more complex subcellular regulation of NO generation has been described through interaction of eNOS with specific proteins involved with trafficking within the cell. One such protein, eNOS traffic-inducing protein (NOSTRIN), was first identified by our group (W.M.E.) in a yeast 2-hybrid approach.13Zimmermann K. Opitz N. Dedio J. Renne C. Muller-Esterl W. Oess S. NOSTRIN: a protein modulating nitric oxide release and subcellular distribution of endothelial nitric oxide synthase.Proc Natl Acad Sci U S A. 2002; 99: 17167-17172Crossref PubMed Scopus (142) Google Scholar Subsequent work has demonstrated an interaction and colocalization between eNOS and NOSTRIN in cultured vascular endothelial cells and eNOS-expressing cell lines and that NOSTRIN overexpression results in a redistribution of eNOS into intracellular vesicle-like structures while significantly inhibiting NO release.13Zimmermann K. Opitz N. Dedio J. Renne C. Muller-Esterl W. Oess S. NOSTRIN: a protein modulating nitric oxide release and subcellular distribution of endothelial nitric oxide synthase.Proc Natl Acad Sci U S A. 2002; 99: 17167-17172Crossref PubMed Scopus (142) Google Scholar Similarly, other eNOS-interacting proteins such as NOSIP may be instrumental in displacing eNOS from plasma membrane caveola with a consequent inhibition of NO generation.14Dedio J. Konig P. Wohlfart P. Schroeder C. Kummer W. Muller-Esterl W. NOSIP, a novel modulator of endothelial nitric oxide synthase activity.FASEB J. 2001; 15: 79-89Crossref PubMed Scopus (144) Google ScholarIn this study, we assessed whether the greater severity of portal hypertension previously described in patients with inflammatory AH compared with patients with cirrhosis alone is associated with differences in gene expression for the NOS-regulatory proteins caveolin-1, NOSIP, and NOSTRIN. To this end, we directed our study to the following areas: first, the determination of hepatic eNOS gene and protein expression and eNOS activity in patients with alcoholic cirrhosis with and without inflammation; second, the measurement of hepatic gene expression of caveolin-1, NOSIP, and NOSTRIN as well as protein expression of NOSTRIN and determination of any association with eNOS activity; and third, a search for alternative splicing products of NOSTRIN in diseased tissues.Materials and MethodsPatientsWe included patients in our study if they had a clearly documented history of alcohol consumption of >80 g alcohol/day for men and >60 g/day for women up until the time of admission. Admission to hospital was precipitated by decompensation, defined by the development of progressive jaundice, derangement of coagulation and liver function tests, or/and the development or worsening of ascites. All eligible patients underwent transjugular liver biopsy and portal pressure assessment as clinically indicated, and a sample of liver biopsy tissue was snap frozen in liquid nitrogen for further evaluation. Patients were excluded if they were 75 years of age and had any of the following: renal dysfunction (plasma creatinine >130 μmol/L), severe cardiovascular or cerebrovascular disease, active or latent infection, hepatic or extrahepatic malignancy, and treatment with vasoactive agents or corticosteroid therapy in the prior month. The local committee on the ethics of human research granted ethical approval, and all patients gave written informed consent. Liver tissue was assessed by an independent histopathologist for evidence of alcoholic cirrhosis, and further criteria applied (balloon degeneration of hepatocytes, Mallory bodies, neutrophilic infiltrates, and apoptotic acidophilic bodies) to define a subgroup of patients with AH.15MacSween R.N. Burt A.D. Histologic spectrum of alcoholic liver disease.Semin Liver Dis. 1986; 6: 221-232Crossref PubMed Scopus (201) Google Scholar Liver tissue was also obtained from 6 additional patients undergoing hepatic resection for metastatic disease of a colonic primary lesion, who had no significant history of alcohol intake and no evidence of liver disease or derangement of liver function tests. Histologic inclusion criteria of the 6 selected specimens included a clear resection margin with no evidence of cellular atypia and no evidence of cirrhosis.Hepatic Wedge Pressure MeasurementsAll wedge pressure assessments were performed after an overnight fast, with patients supine for at least 1 hour prior to the procedure, and controlled sedation was given using midazolam (mean dose of 3 mg; Phoenix Pharma Ltd., Gloucester, United Kingdom). For measurement of hepatic venous pressure gradient (HVPG), the right hepatic vein was cannulated under fluoroscopic screening (Toshiba Spot Film Device Model: SA-900U; Tochigi-ken, Japan), and wedged pressure measurements were assessed in triplicate in at least 2 radicals after injection of 2 mL contrast medium (Iohexol [Omnipaque]; Amersham Health, Little Chalfont, United Kingdom). Wedged hepatic venous pressure (WHVP) and free hepatic venous pressure (FHVP) measurements were recorded via pressure transducer sets (Medex Medical, Rossendale, Lancashire, United Kingdom) on a Hewlett Packard monitor (Model 86S; HP, Palo Alto, CA). HVPG was calculated as the difference between WHVP and FHVP. The coefficient of variation of the measurements of HVPG within a given patient was 3% (0.42) for the study group. Blood pressure and heart rate were measured noninvasively by standard methods.Quantitative Real-Time Polymerase Chain ReactionmRNA and protein was isolated from Hep3B cells and liver tissue using the TRI-Reagent (Sigma, Taufkirchen, Germany) following the manufacturer's instructions. From the messenger RNA (mRNA), complementary DNA (cDNA) was synthesized using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA) according to manufacturer's recommendations. Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) assays were developed using the TaqMan technology on an iCycler detection system (Bio-Rad). Assay mix of primers and TaqMan MGB probes (FAM dye-labelled) for eNOS, caveolin, NOSTRINβ (ie, the N-terminally truncated form of NOSTRIN, see below), and control ubiquitin C spanning exon-exon junctions to exclude detection of genomic DNA were from Applied Biosystems (Foster City, CA). To detect NOSTRINα (the full-length form), an "assay-on-design" (Applied Biosystems) was used with the probe spanning the junction of exon-2 to exon-3 of the NOSTRIN sequence. Up to 4 μL whole cDNA from cells were used per 50 μL-qRT-PCR reaction mixture. In case of whole liver cDNA, each cDNA sample was split into 6 equal aliquots and analyzed for target genes and endogenous control. Duplicate cycle threshold (CT) values were normalized against the internal control ubiquitin C and analyzed using the ΔCT method. The relative expression levels were calculated from the normalized mean CT values.ImmunohistochemistryLiver sections were embedded either in Tissue-Tek (Sakura Finetek, Zoeterwoude, the Netherlands) for cryoconservation or paraffin and then stained by standard H&E and immunohistochemical methods. Briefly, paraffin-embedded sections, pretreated with xylene, were microwaved in citrate buffer for 20 minutes to optimize antigen retrieval and were treated with monoclonal antibody to human eNOS (Transduction Laboratories, Lexington, KY). Tissue-Tek-embedded 12-μm liver sections were fixed in 4% (wt/vol) paraformaldehyde. A poly- or monoclonal anti-human NOSTRIN (raised against a fusion construct of glutathione s-transferase and NOSTRIN242-506 (GST-NOSTRIN242-506); polyclonal: rabbit anti-NOSTRIN [AS532]; monoclonal: by Nanotools, Teningen, Germany) was used for NOSTRIN staining. For control, the antibodies were blocked with GST-NOSTRIN prior to the staining.Cell Culture and TransfectionHuman hepatocellular carcinoma (Hep3B) cells were cultured in Dulbecco's modified Eagle medium (DMEM)/10% fetal calf serum (FCS) with or without 1 μmol/L retinoic acid (RA) for up to 6 days. Chinese hamster ovary (CHO) cells stably expressing eNOS (CHO-eNOS) were cultured in DMEM/10% FCS containing 200 nmol/L methotrexate. Transient transfections with pcDNA-3.1B-NOSTRINα and pcDNA-3.1B-NOSTRINβ were performed with Nanofectin (PAA, Coelbe, Germany) according to the manufacturer's instructions. Cells on coverslips were fixed in methanol 20 hours posttransfection, treated with monoclonal anti-NOSTRIN, and analyzed by confocal laser-scanning microscopy.Western BlotsProtein fractions of whole liver samples were isolated with TRI-Reagent according to the manufacturer's instructions. These fractions as well as whole cells were dissolved in Laemmli buffer (63 mmol/L Tris-HCl, pH 6.8, 2.5% SDS, 5% glycerol, 5% β-mercaptoethanol, 0.005% bromophenol blue), subjected to SDS-PAGE, and transferred to polyvinylidene difluoride (PVDF) membranes, which were then probed with mouse monoclonal anti-human lysosome-associated membrane protein-1 (Lamp-1) (Transduction Laboratories) and monoclonal anti-NOSTRIN.CoimmunoprecipitationHuman liver was frozen in liquid nitrogen, ground, and lysed in N-octylglycoside (OG) buffer (50 mmol/L Tris HCl, pH 7.4; 2 mmol/L EDTA; 1 mmol/L EGTA; 50 mmol/L NaF; 150 mmol/L NaCl; 1 mmol/L Na3VO4; 10 mmol/L Na4P2O7; 1 mmol/L DTT; 60 mmol/L N-octylglycoside; protease inhibitor cocktail [Roche Diagnostics GmbH, Mannheim, Germany]). Coimmunoprecipitation was performed using polyclonal rabbit anti-NOSTRIN (AS532). Western blots of co-immunoprecipitates and lysates were probed with mouse anti-eNOS, anti-caveolin-1 (Transduction Laboratories), and anti-NOSTRIN (see above).Quantification of Tissue eNOS ActivityThe method used for quantification of tissue eNOS activity was a variation of the 3H-arginine-citrulline conversion assay16Bredt D.S. Snyder S.H. Nitric oxide mediates glutamate-linked enhancement of cGMP levels in the cerebellum.Proc Natl Acad Sci U S A. 1989; 86: 9030-9033Crossref PubMed Scopus (1747) Google Scholar, 17Bredt D.S. Snyder S.H. Isolation of nitric oxide synthetase, a calmodulin-requiring enzyme.Proc Natl Acad Sci U S A. 1990; 87: 682-685Crossref PubMed Scopus (3114) Google Scholar modified for use with liver tissue. Briefly, snap-frozen liver tissue was homogenized in ice-cold HEPES buffer (20 mmol/L, pH 7.4) containing EDTA (1 mmol/L), sucrose (250 mmol/L), valine (60 mmol/L), protease inhibitor cocktail (at 1 mL/20 g tissue, containing 4-(2-aminoethyl)benzenesulfonyl fluoride [AEBSF], pepstatinA, E-64, bestatin, leupeptin, and aprotinin; Sigma-Aldrich Co, Gillingham, United Kingdom), and phenylmethylsulfonyl fluoride (3.4 mg/mL). This was centrifuged (1000g, 10 minutes, 4°C) and the supernatant retained and measured for protein content using the Biuret method.18Gornall A.G. Determination of serum proteins by means of the Biuret reaction.J Biol Chem. 1949; 177: 751-766Abstract Full Text PDF PubMed Google Scholar For NOS activity determination, 5 μL supernatant was incubated with 40 μL reaction medium containing the following: Tris-HCL buffer (30 mmol/L, pH 7.4); NADPH (1.25 mmol/L); 3H-arginine (10 μCi/mL, Amersham Health, Buckinghamshire, United Kingdom); norvaline (5 mmol/L); and either CaCl2 (400 μmol/L) or EGTA (600 μmol/L) at 30°C for 30 minutes and stopped with 1 mL ice-cold citrate buffer (50 mmol/L, pH5) containing EDTA (1 mmol/L). The arginine/citrulline ratio was determined by separating the amino acid components using thin layer chromatography on silica plates (Kieselgel 60; Merck, Darmstadt, Germany). Nontritiated amino acids were added to aid spot detection and the components separated using a running mixture of CHCl3/MeOH/NH4OH/H2O at a ratio of 10:45:20:10. The individual spots were removed, and the scintillation activity was measured (Ultima Gold scintillant; Perkin Elmer, Boston, MA), using a Tri-Carb 2100TR, liquid scintillation analyser (Packard Biosciences, Berks, United Kingdom). Activity was expressed as micromoles citrulline/mg protein/h. All chemicals and reagents were supplied by the Sigma Chemical Company (Poole, United Kingdom) unless otherwise stated.ResultsPatientsBiopsy specimens from 20 decompensated alcoholic cirrhotic patients were included, with 10 of these fulfilling histologic and clinical criteria for alcoholic hepatitis superimposed on cirrhosis (AH+C). All the AH+C biopsy specimens had a moderate to severe grading of necroinflammation using a parallel grading system of scoring suggested for nonalcoholic steatohepatitis.19Brunt E.M. Janney C.G. Di Bisceglie A.M. Neuschwander-Tetri B.A. Bacon B.R. Nonalcoholic steatohepatitis: a proposal for grading and staging the histological lesions.Am J Gastroenterol. 1999; 94: 2467-2474Crossref PubMed Scopus (3067) Google ScholarPatients with AH+C were of similar age when compared with patients with alcoholic cirrhosis alone (AC), but, expectedly, they had significantly higher Pugh scores (P = .002). Age-matched patients with no biochemical or histologic manifestation of liver disease were used as controls. In addition to histologic criteria, all AH+C patients had acute severe alcoholic hepatitis as evidenced by a mean Maddrey discriminant function score of 41 ± 820Maddrey W.C. Boitnott J.K. Bedine M.S. Weber Jr, F.L. Mezey E. White Jr, R.I. Corticosteroid therapy of alcoholic hepatitis.Gastroenterology. 1978; 75: 193-199PubMed Scopus (685) Google Scholar and as reflected in the significantly elevated plasma bilirubin levels and prothrombin times compared with AC patients, as summarized in Table 1. There were, however, no statistically significant differences in albumin or serum aminotransferase levels between the alcoholic liver disease patient groups. As anticipated from their histologic grading, AH+C patients had significantly higher inflammatory indices compared with AC patients, evidenced by higher plasma C-reactive protein (P = .007) and higher systemic inflammatory response syndrome components21Bone R.C. Sibbald W.J. Sprung C.L. The ACCP-SCCM consensus conference on sepsis and organ failure.Chest. 1992; 101: 1481-1483Crossref PubMed Scopus (599) Google Scholar (P = .001). Moreover, the heightened state of inflammation in the AH+C patients was also notable from their significantly greater plasma tumor necrosis factor receptor (TNF-RI) (9 ± 0.9 vs 4 ± 0.8 ng/mL, respectively; P = .004) and TNF-RII levels (18 ± 1.3 vs 7.6 ± 1.5 ng/mL, respectively; P < .001).Table 1Patient CharacteristicsAH+C (n = 10)AC (n = 10)P valuesAge, yr53 ± 449 ± 3.4Sex4 F, 6 M2 F, 8 M—Pugh score11 ± 0.48 ± 0.6.002PT, s (range, 11−14)16 ± 213 ± 0.3.04Bilirubin, μmol/L (5–17)313 ± 11176 ± 27.02ALT, U/L (5–40)96 ± 4437 ± 7.2Albumin, g/L, (35−50)26 ± 129 ± 2.2CRP, mg/L (0−5)43 ± 813 ± 2.007SIRS, (0)1.4 ± 0.20.3 ± 0.2.001WBC, ×109/L (3–10)15.3 ± 38.8 ± 1.1.07NOTE. Physiologic ranges are indicated in parentheses.PT, prothrombin time; ALT, alanine aminotransferase; CRP, C-reactive protein; SIRS, systemic inflammatory response syndrome; WBC, white blood cell count. Open table in a new tab HemodynamicsPatients with AH+C had significantly higher HVPG compared with the cirrhotic patients (23 ± 2 vs 15 ± 0.6 mm Hg, respectively; P = .005). There were, however, no significant differences noted in mean arterial pressures (P = .7) or heart rate (P = .4) between the 2 groups.mRNA QuantificationUsing quantitative RT-PCR, we found that eNOS mRNA levels in the liver samples of inflammatory AH+C patients were significantly increased (as denoted by markedly lower CT values) compared with AC patients (7.2 ± 0.4 vs 9.8 ± 0.4, respectively, P = .001) and noncirrhotic patients (11 ± 1.8, P = .02) (Figure 1A; note that values are given as 1/CT). No significant difference was found between eNOS mRNA expression levels in cirrhotic livers without inflammation and normal livers (P = .25). Caveolin-1 mRNA expression levels were similarly significantly higher in AH+C compared with cirrhotic livers (4.6 ± 0.3 vs 6.3 ± 0.4, respectively, P = .007) and when compared with normal liver (9.7 ± 2.1, P = .02) (Figure 1B). There were, however, no significant differences observed in NOSIP mRNA expression levels between the alcoholic patient groups and normal patients (P = .3 and P = .5, respectively) (data not shown). In contrast, NOSTRIN mRNA expression levels were significantly higher in liver samples of AH+C patients compared with AC patients (2.3 ± 0.3 vs 3.4 ± 0.2, respectively, P = .009; Figure 1C). Liver samples of AC patients also had significantly higher NOSTRIN mRNA expression levels compared with normal tissue (6.8 ± 2, P = .04).Of interest, we found a second mRNA species hybridizing with NOSTRIN-specific probes representing a novel isoform of NOSTRIN that lacks exon-2. The shortened isoform was designated NOSTRINβ, whereas we will from hereon refer to full-length NOSTRIN as NOSTRINα. Importantly, NOSTRINβ mRNA was significantly elevated in AH+C patients, as compared with those without inflammation (15.1 ± 0.7 vs 19.6 ± 1.7, P = .04), whereas it was not detected at all in normal tissue (Figure 1D). Sequence analysis of the NOSTRINβ cDNA revealed that skipping of exon-2 generates a premature stop codon in exon-3 such that an internal methionine codon present in exon-4 is used for alternative transcription initiation at the NOSTRINβ mRNA. This results in a shortened isoform lacking the first 78 amino acid residues at the N-terminus of NOSTRIN (see below).Assessment of eNOS Protein Expression and ActivityImmunostaining of liver tissue specimens with anti-human eNOS demonstrated eNOS expression in both AH+C and AC biopsy specimens, albeit this method was semiquantitative and thus did not allow a clear difference to be defined between the 2 alcoholic liver disease groups (Figure 2A, a and b). Total NOS activity was significantly lower in AH+C patients compared with AC patients (38.5 ± 12.6 vs 91.4 ± 31.8 nmole citrulline/h/mg liver tissue, p=0.04). eNOS activity was calculated following Ca2+ chelation (total minus Ca2+-independent activity) and also demonstrated significantly lower activity in AH+C patients (23 ± 6.4 vs 46 ± 13 nmol citrulline/h/mg liver tissue, respectively, P = .04). The eNOS activity of AC patients was slightly reduced compared with normal control tissue, but this did not prove to be statistically significant (data not shown). The HVPG was also found to have a significant association with hepatic tissue eNOS activity (r = −0.64; P = .01; Figure 2B).Figure 2eNOS expression and activity in liver tissue. (A) Immunostaining of paraffin-embedded liver biopsy sections from alcoholic hepatitis (AH+C) (a) and cirrhosis alone (AC) patients (b) using monoclonal anti-human eNOS. The enzyme is present in biopsy specimens from both AH+C patients (panel A) and AC (panel B). Semiquantitative assessment of the staining intensity did not allow a clear difference to be defined in eNOS expression between AH+C and AC patients (arrows). Original magnification, ×40. (B) Graph suggests a statistically significant correlation between reduced eNOS generation and high portal pressure by demonstrating that eNOS tissue activity is inversely correlated with the severity of hepatic venous pressure gradient: r = −0.64 and P = .01.View Large Image Figure ViewerDownload Hi-res image Download (PPT)NOSTRIN Protein Expression and LocalizationTo analyze for expression of NOSTRIN protein, Western blots were performed on lysates of human liver tissue specimens with a monoclonal antibody binding to the C-terminal portion that is shared by NOSTRINα and β (Figure 3A). In Western blots, the 2 NOSTRIN isoforms clearly differ by their size, as demonstrated by recombinant expression of the proteins in CHO cells. NOSTRINα is detected at 58 kilodaltons and NOSTRINβ at 50 kilodaltons (Figure 3B), and, thus, they can be easily distinguished. In the liver samples, NOSTRINα protein expression was readily detectable at the expected size in all patient samples analyzed (
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