Prof L Leong

MA Ng

Hafid Narayan Bm

Bsc Mrcp

MS Paulene Quinn

MD FRCP Iain B Squire

PhD Joan E Davies

Proteinase 3 and prognosis of patients with acute myocardial infarction

Keywords: Myocardial infarction, peptides, proteinase 3, myeloperoxidase, neutrophil, prognosis

Background

A multimarker approach may be useful for risk stratification in AMI patients, particularly utilising pathways that are pathophysiologically distinct.

Aim

Our aim was to assess the prognostic value of Proteinase 3 in patients post acute myocardial infarction (AMI). We compared the prognostic value of Proteinase 3, an inflammatory marker to an established marker N-terminal pro-B-type natriuretic peptide (NT-proBNP) post-AMI.

Method

We recruited 900 consecutive post-AMI patients (700 men, age 64.6 12.4 years) in a prospective study with follow-up over 347[0-764] days. Results Plasma Proteinase 3 was significantly higher in patients who died (666.2 [226.8-4035.5] ng/ml, P<0.001) or were readmitted with heart failure (598 [231.6-1803.9] ng/ml, P<0.004) compared to event free survivors (486.9 [29.3-3118.2] ng/ml). Using Cox modelling log10 Proteinase 3 (HR 3.80) and log10NT-proBNP (HR 2.51) were significant independent predictors of death or heart failure. When patients were stratified by plasma NTproBNP (median 1023 pmol/L), Proteinase 3 gave additional predictive value for death or heart failure, in both the patients in whom NTproBNP level was above the median (log rank for trend 12.54, p<0.0004) and those with NTproBNP level below the median (log rank for trend 3.83, p<0.05). Neither marker predicted recurrent AMI. Conclusion This is the first report showing a potential role for the serine protease Proteinase 3 in determining mortality and incidence of heart failure following AMI, independent of established conventional risk factors. Proteinase 3 may represent a clinically useful marker of prognosis after an AMI as part of a multimarker strategy.

Introduction

Atherosclerosis is associated with inflammatory changes in the blood vessel wall [START_REF] Libby | Pathophysiology of coronary artery disease[END_REF], including macrophage and lymphocyte infiltration. There is an association between neutrophil count and the risk of future acute coronary syndromes [START_REF] Friedman | The leukocyte count as a predictor of myocardial infarction[END_REF], and recent evidence has documented elevated neutrophil activity in acute coronary syndromes [START_REF] Naruko | Neutrophil infiltration of culprit lesions in acute coronary syndromes[END_REF][START_REF] Buffon | Widespread coronary inflammation in unstable angina[END_REF]. Despite their role in plaque destabilisation and acute coronary syndromes, it is not known whether neutrophil activation, with degranulation of their proteolytic enzymes might play a role in development of chronic heart failure and the risk of mortality following an acute myocardial infarction (AMI). Myeloperoxidase (MPO), an enzyme present within the azurophilic granules of neutrophils, may stimulate plaque destabilisation by activation of metalloproteinases [START_REF] Fu | Hypochlorous acid oxygenates the cysteine switch domain of pro-matrilysin (MMP-7). A mechanism for matrix metalloproteinase activation and atherosclerotic plaque rupture by myeloperoxidase[END_REF]. Plasma levels of MPO are raised in coronary artery disease [START_REF] Zhang | Association between myeloperoxidase levels and risk of coronary artery disease[END_REF], and predict outcome of patients presenting with chest pain [START_REF] Brennan | Prognostic value of myeloperoxidase in patients with chest pain[END_REF] and acute coronary syndromes [START_REF] Baldus | Myeloperoxidase serum levels predict risk in patients with acute coronary syndromes[END_REF][START_REF] Khan | Myeloperoxidase aids prognostication together with N-terminal pro-B-type natriuretic peptide in high-risk patients with acute ST elevation myocardial infarction[END_REF]. Proteinase 3 (PR3) is another major component of neutrophil azurophilic granules, together with other serine proteases such as elastase and cathepsin G [START_REF] Pham | Neutrophil serine proteases: specific regulators of inflammation[END_REF]. Azurophilic granules are released in response to powerful stimuli to neutrophils, whereas secretory granules (which also contain PR3 [START_REF] Witko-Sarsat | Presence of proteinase 3 in secretory vesicles: evidence of a novel, highly mobilizable intracellular pool distinct from azurophil granules[END_REF]) are released with weaker stimuli. PR3 may also be expressed on endothelial cells [START_REF] Mayet | Human endothelial cells express proteinase 3, the target antigen of anticytoplasmic antibodies in Wegener's granulomatosis[END_REF]. Neutrophil serine proteases may degrade extracellular matrix; in addition, PR3 has other deleterious effects which may be relevant to a role in pathogenesis of vasculitis in Wegener's granulomatosis and potentially in prognosis of patients post-AMI. These include induction of apoptosis through a caspase-like activity on endothelial cells [START_REF] Pendergraft | Proteinase 3 sidesteps caspases and cleaves p21(Waf1/Cip1/Sdi1) to induce endothelial cell apoptosis[END_REF], releasing activated TNF from its nascent membrane bound precursor form [14,[START_REF] Coeshott | Converting enzyme-independent release of tumor necrosis factor alpha and IL-1beta from a stimulated human monocytic cell line in the presence of activated neutrophils or purified proteinase 3[END_REF], activation of the pro-inflammatory mediators interleukin-1β [START_REF] Coeshott | Converting enzyme-independent release of tumor necrosis factor alpha and IL-1beta from a stimulated human monocytic cell line in the presence of activated neutrophils or purified proteinase 3[END_REF] and interleukin-18 [START_REF] Sugawara | Neutrophil proteinase 3-mediated induction of bioactive IL-18 secretion by human oral epithelial cells[END_REF] and generation of angiotensin I and II from angiotensinogen [START_REF] Ramaha | Release and degradation of angiotensin I and angiotensin II from angiotensinogen by neutrophil serine proteinases[END_REF]. Although PR3 and autoantibodies to it (anti-neutrophil cytoplasmic antibodies or ANCA) are prime candidates for pathogenesis of vasculitis and tissue damage in the relatively uncommon autoimmune condition of Wegener's granulomatosis, the association of PR3 with morbidity and mortality in common medical vascular presentations such as AMI has never been examined. PR3 is rapidly inactivated by irreversible binding to -1-antitrypsin (SERPIN A1) [START_REF] Baslund | Measurements of proteinase 3 and its complexes with alpha 1-proteinase inhibitor and anti-neutrophil cytoplasm antibodies (ANCA) in plasma[END_REF][START_REF] Duranton | Inhibition of proteinase 3 by alpha 1-antitrypsin in vitro predicts very fast inhibition in vivo[END_REF] where the serine protease reacts with the reactive centre loop of the serpin, generating a covalently bound intermediate resulting in the distortion and inactivation of the protease [20]. Thus, little free PR3 exists in plasma and it is mainly present as a PR3-SERPIN A1 complex [START_REF] Baslund | Measurements of proteinase 3 and its complexes with alpha 1-proteinase inhibitor and anti-neutrophil cytoplasm antibodies (ANCA) in plasma[END_REF]. We hypothesised that measurement of such PR3-SERPIN A1 complexes would provide prognostic information on heart failure and mortality post-AMI, even when used in conjunction with recognised markers of prognosis such as B-type natriuretic peptide or its precursor (N-terminal proB-type natriuretic peptide or NTproBNP) in order to establish a role for neutrophil activation in determining these adverse outcomes. Since PR3 is easily mobilised from secretory granules compared to azurophilic granules, it may also be a better marker of neutrophil activation than MPO.

Methods

Study population

We recruited 900 consecutive patients with acute myocardial infarction admitted to the Coronary Care Unit of Leicester Royal Infirmary between 1st September 2003 and 31st July 2007. Written informed consent was obtained from patients and the study complied with the Declaration of Helsinki and was approved by the local ethics committee. AMI was diagnosed if a patient had a plasma creatine kinase-MB elevation greater than twice normal or cardiac troponin I level >0.1 ng/mL with at least one of the following, chest pain lasting >20 minutes or diagnostic serial electrocardiographic (ECG) changes consisting of new A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

pathological Q waves or ST-segment and T-wave changes. Exclusion criteria included malignancy or recent surgery (within one month). Hypertension was defined as a blood pressure measurement of greater that 140/90 on 2 or more occasions or if the patient was on medication for hypertension. Hypercholesterolaemia was defined as a total cholesterol of greater than 5.0 mmol/L, or treatment with a statin. Diabetes was defined as a random plasma glucose of 11.1mmol/L, fasting plasma glucose of 7mmol/L or a history of diabetes on treatment.

Plasma samples

Blood samples were drawn at 2-5 days after the onset of chest pain for determination of plasma PR3-SERPIN A1 and NTproBNP. After 15 minutes bed rest, 20mL blood was collected into tubes containing EDTA and aprotinin. All plasma was stored at -80°C until assayed, blind to clinical details, in a single batch.

Echocardiography

Transthoracic echocardiography was performed in patients using a Sonos 5500 instrument (Philips Medical Systems, Reigate, UK), at 3-5 days post-admission. A 16-segment left ventricular wall motion index (LVWMI) based on the American Society of Echocardiography mode was derived by scoring each LV segment (1=normal, 2=hypokinesis, 3=akinesis and 4=dyskinesis (Paradoxical Motion), and dividing the total by the number of segments scored. Left ventricular ejection fraction (LVEF) was calculated using the biplane method of discs formula [START_REF] Schiller | Recommendations for quantitation of the left ventricle by two-dimensional echocardiography[END_REF]. Impaired LV systolic function was defined as an EF<40% or a LVWMI >1.8.

NTproBNP assay

Plasma NTproBNP assay was measured using a non-competitive immunoluminometric assay as previously published [START_REF] Omland | N-terminal pro-B-type natriuretic peptide and long-term mortality in acute coronary syndromes[END_REF] . Samples or NTproBNP standards (10μL) were incubated in ELISA plate wells coated with mouse monoclonal IgG directed to the C-terminal of NTproBNP. Detection was with biotinylated rabbit N-terminal antibody followed by methylacridinium ester (MAE)-labelled streptavidin on a MLX plate luminometer (Dynex Technologies Ltd., Worthing, UK). The lower limit of detection was 0.3 pmol/L. This highly specific assay shows no cross-reactivity with atrial natriuretic peptide, BNP, or C-type natriuretic peptide.

PR3-SERPIN A1 assay

Plasma PR3-SERPIN A1 was measured using a non-competitive immuno-luminometric assay modified from a previously published method [START_REF] Baslund | Measurements of proteinase 3 and its complexes with alpha 1-proteinase inhibitor and anti-neutrophil cytoplasm antibodies (ANCA) in plasma[END_REF]. ELISA plate wells were coated with 200ng of mouse monoclonal antibody to PR3 (clone 1B10, IgG 2a , HyTest Ltd., Turku, Finland). Standards were produced by incubating PR3 with a 20 fold molar excess of SERPIN A1 (both from Sigma Aldrich Co. Ltd., Gillingham, UK). Samples or standards (10μL) were incubated with assay buffer (100μL) in the coated wells for 24 h at 4ºC. Following washes, a specific SERPIN A1 rabbit antibody (Dako UK Ltd., Ely, UK) was pipetted into the wells (20ng/100μL) and incubated for 3 h with shaking at 250 Hz at room temperature. Following washes, a goat biotinylated anti-rabbit IgG (Rockland Immunochemicals Inc., Gilbertsville, PA, USA, previously pre-adsorbed with human, rabbit, mouse serum proteins) at a dilution of 1:100000 was incubated within the wells for 1 h, followed by MAE-labelled streptavidin for another 11⁄2 hours. Chemiluminescence was elicited with sequential injections of H 2 O 2 in nitric acid, followed by sodium hydroxide containing cetyl trimethylammonium bromide, as described [START_REF] Omland | N-terminal pro-B-type natriuretic peptide and long-term mortality in acute coronary syndromes[END_REF]. Intra and inter-assay coefficients of variation were found to be less than 10%. A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Myeloperoxidase (MPO) assay

This was based on a 2 site non-competitive immuno-luminometric assay employed an ELISA plate immobilised monoclonal MPO antibody (100ng/100 L, Research Diagnostics Inc., New Jersey, USA) and a rabbit polyclonal antibody (50ng/100 L, Merck BioSciences, Nottingham, UK), as previously described [START_REF] Ng | Myeloperoxidase and C-reactive protein augment the specificity of B-type natriuretic peptide in community screening for systolic heart failure[END_REF]. Standards and 10 μL of plasma were incubated in plates overnight, before washing and detection using goat biotinylated antirabbit IgG and streptavidin-MAE, as described for the PR3-SERPIN A1 assay. The MPO assays had inter-and intra-assay coefficients of variation <10%, with a lower limit of detection of 0.3 ng/ml.

End points

The primary endpoint was the combination of all-cause mortality and hospitalisation for heart failure. Secondary endpoints included the occurrence of further myocardial infarction. Hospitalization for heart failure was defined as a hospital admission for which heart failure was the primary reason, requiring treatment with high dose diuretics, inotropes, or intravenous nitrate. Deaths were validated by reviewing the hospital record management systems and the Office of National Statistics Registry. Heart failure episodes were identified through the hospital record management systems and validated by contacting each patient. There was a minimum 60-day follow-up of all surviving patients.

Statistical analysis

Statistical analyses were performed on SPSS Version 14.0 (SPSS Inc, Chicago, Illinois). Continuous variables were compared using the Mann Whitney U test. ANOVA with the Bonferroni correction for multiple comparisons was employed when more than 2 groups were compared. Correlation analysis employed Spearman's rho, and binary logistic regression and Cox proportional hazard analyses were used to develop models in order to test the independent predictive power for factors and variables. Plasma levels of NTproBNP, MPO and PR3-SERPIN A1 were normalised by log transformation. Thus, odds ratios and hazard ratios refer to a tenfold rise in the levels of these markers. Kaplan-Meier survival analysis was used to assess dichotomised values of NTproBNP and PR3-SERPIN A1 and the log rank test for used for comparison of groups. Receiver-operating characteristic (ROC) curves were generated and the area under the curves (AUC) was calculated to assess the accuracy of predictors [START_REF] Hanley | A method of comparing the areas under receiver operating characteristic curves derived from the same cases[END_REF]. A p value of less than 0.05 was deemed to be statistically significant. All authors had full access to the data and take responsibility for its integrity and accuracy of the analysis. All authors have read and agree to the manuscript as written. Roles of authors include:-design of study and assay construction LN; recruitment of patients SQK; echocardiography analysis SQK and JED; assays LN, PQ; analysis of data LN, SQK, IBS, HN; manuscript preparation, all authors.

Results

Patient characteristics

The demographic features of the patient population are shown in Table 1. Median length of follow-up was 347 days with a range of 0-764 days. No patient was lost to follow-up. The index AMI was classed as ST-elevation MI (STEMI) in 777 of the patients, 529 (68.1%) of whom received thrombolytic therapy. Echocardiographic data were available for 622 (69.1%) of the patients during the index admission. During follow-up, 92 (10.2%) of the 900 patients died, 66 (7.3%) experienced readmission with heart failure, 143 (15.9%) patients experienced either end-point and 62 (6.9%) experienced hospitalisation with recurrent AMI. Plasma PR3-SERPIN A1, NTproBNP and MPO levels Plasma levels of PR3-SERPIN A1 in patients with AMI ranged from 29.3-4035.5 ng/ml with a median of 504.6 ng/ml, and were elevated compared to the established normal range (median [range] 350 [110-580] ng/ml). PR3-SERPIN A1 was significantly higher in patients who died (666.2 [226.8-4035.5] ng/ml, P<0.001 using Bonferroni's correction) or were readmitted with heart failure (598 [231.6-1803.9] ng/ml, P<0.004) compared to event free survivors (486.9 [29.3-3118.2] ng/ml). In contrast, levels of PR3-SERPIN A1 in those readmitted with recurrent AMI (482.5 [168.9-1753.9] ng/ml) were similar to those in event free survivors. PR3-SERPIN A1 levels were similar in males and females, and in those with or without previous histories of hypertension, angina, AMI or heart failure, but were slightly elevated in those with diabetes (547.0 [76.9-4035.5] ng/ml) vs. non-diabetic patients (495.1 [29.3-3118.2] ng/ml, P<0.018). Plasma PR3-SERPIN A1 levels were modestly correlated with plasma glucose (r s = 0.08, p< 0.03), log creatinine (r s = 0.13, p< 0.001), Killip class (r s = 0.10, p< 0.002), NTproBNP (r s =0.25, p< 0.001) and creatine kinase (r s =0.13, p< 0.001) but had no relationship to age, site of infarction, presence or absence of ST elevation and whether thrombolysis was administered or not. In confirmation of previous work, plasma NTproBNP was higher in patients who died (5955. There was a modest correlation of Plasma PR3-SERPIN A1 with impaired LV systolic function (r s = 0.16, p= 0.001) on the index admission (as defined by EF<40% or LVWMI>1.8 on echocardiography) with a stronger relationship demonstrated for NTproBNP (r s = 0.35, p= 0.001). Plasma MPO was weakly correlated to PR3-SERPIN A1 and NTproBNP (r s = 0.11 and 0.12 respectively, p= 0.002) but not to LV systolic function.

Primary Endpoints: PR3-SERPIN A1 and NTproBNP as predictors of death or heart failure

Binary logistic models were developed for prediction of death or heart failure, using the following clinical and demographic variables:-age, gender, past history of AMI, hypertension or diabetes, Killip Class>1, thrombolytic use, log creatinine, NTproBNP, MPO and PR3-SERPIN A1. Independent predictors are reported in Table 2 and include NTproBNP and PR3-SERPIN A1 as significant predictor variables. The Nagelkerke r 2 was 0.35 suggesting a good fit of the model. The receiver-operating-characteristic curve for the predictive value of PR3-SERPIN A1 for the primary endpoint yielded an area under the curve (AUC) of 0.65 (95% CI: 0.60-0.70, p<0.001); for NTproBNP the AUC was 0.78 (95% CI: 0.74-0.83, p<0.001). The logistic model combining the 2 markers yielded an AUC of 0.84 (95% CI: 0.80-0.88, p<0.001), which exceeded that of either peptide alone (p<0.003 by method of Hanley and McNeil [24]). Cox proportional hazards modelling confirmed these findings, identifying the same independent predictors of death or heart failure apart from gender (Table 3). In both logistic and Cox models, plasma MPO was not an independent predictor of death or heart failure. Kaplan-Meier survival analysis demonstrated a significantly better clinical outcome in patients with PR3-SERPIN A1 below the median (504.6 ng/ml) compared with those with PR3-SERPIN A1 above the median (log rank 28.15, p<0.0001, figure 1). The event free survival curves diverge early and continue to diverge even after 2 years. This was also true for NTproBNP (log rank 64.72, p<0.0001). When patients were stratified by plasma NTproBNP (median 1023 pmol/L), PR3-SERPIN A1 gave additional predictive value for death or heart failure, in both the patients in whom NTproBNP level was the median (log rank for trend 12.54 p<0.0004) and those with NTproBNP level below the median (log rank for trend 3.83 p<0.05). Those patients with both markers elevated above their respective medians had higher event rates than those with either marker elevated above median, and these in turn had higher event rates than those with both markers below their respective medians (p<0.001 for all comparisons, figure 2).

Troponin levels were available for 414 patients. Inclusion of troponin in the Cox model for death or heart failure led to the following hazard ratios (and p values) : age (1.07, p<0.001), Killip Class > 1 (1.83, p<0.04), log creatinine (6.05, p<0.06), past history of AMI (1.70, p<0.07), NTproBNP (1.74, p<0.08), troponin (1.18, p=0.3), PR3-SERPIN A1 (2.83, p<0.06).

Echocardiography scans were analysable on 622 subjects, of which 203 showed evidence of LV systolic dysfunction (as defined by EF<40% or LVWMI>1.8). Inclusion of echocardiography evidence of systolic dysfunction into the Cox model for death or heart failure led to the following hazard ratios (and p values) : age (1.04, p<0.001), Killip Class > 1 (2.17, p<0.005), log creatinine (6.55, p<0.01), past history of AMI (1.40, p=0.13), NTproBNP (1.92, p<0.007), LV systolic dysfunction (1.39, p=0.13), PR3-SERPIN A1 (2.88, p<0.02).

Reclassification analyses for predictors of death or heart failure

The GRACE score [START_REF] Eagle | A validated prediction model for all forms of acute coronary syndrome: estimating the risk of 6-month postdischarge death in an international registry[END_REF] is a recognised risk predictor in AMI, enabling patients to be classified into low, intermediate and high risk groups for major adverse cardiac events after an acute coronary syndrome, and incorporates many of the above predictors for death or heart failure e.g. age, history of AMI, elevated plasma creatinine, elevated biomarker levels, Killip class. In this present study, GRACE score was a significant predictor of death or heart failure (hazard ratio 1.03 (95% confidence interval 1.02-1.04, p<0.0005). In a model for predicting death or heart failure using GRACE score, NTproBNP and PR3-SERPIN A1, all 3 remained significant predictors (hazard ratio [95% confidence interval] p values were respectively, GRACE score 1.02 [1.01-1.03], p<0.0005; NTproBNP 2.67 [1.79-3.99], p<0.0005; PR3-SERPIN A1 4.08 [1.89-8.79], p<0.0005).

Reclassification analyses were therefore performed using GRACE score, with the addition of NTproBNP and PR3-SERPIN A1 as described by Pencina et al [START_REF] Pencina | Evaluating the added predictive ability of a new marker: from area under the ROC curve to reclassification and beyond[END_REF]. NTproBNP led to significant up-classification of patients with endpoints and down-classification of event free survivors (Table 4), when added to the GRACE score (p<0.0002). PR3-SERPIN A1 in addition to the GRACE score also led to significant (p<0.02) up-classification of patients with endpoints, although when added to GRACE score with NTproBNP, this upclassification was not significant (p=0.14).

Discussion

This study reports for the first time the prognostic potential of PR3-SERPIN A1 in a large cohort of patients with AMI, recruited prospectively from a single centre. This complex of a serine protease and its inhibitor provided prediction of death or heart failure, independently of established conventional risk factors such as renal function and clinical demographic information and the GRACE score. Moreover this novel prognostic marker complements the information provided by an established marker of poor prognosis post AMI, namely NTproBNP. The use of PR3-SERPIN A1 for independent prediction of death or heart failure together with troponin levels or echocardiographic evidence of systolic dysfunction failed to achieve conventional levels of statistical significance due to the reduced number of subjects with data available for analysis. Reclassification analyses with the GRACE score suggested that PR3-SERPIN A1 had utility, mainly in up-classifying patients with endpoints, in contrast to NTproBNP which up-classified those with endpoints and down-classified those without endpoints. However, adding PR3-SERPIN A1 to models with GRACE score and NTproBNP led to insignificant up-classification of patients with endpoints. The study may have been underpowered to detect significant reclassification in this analysis.

The measurement of PR3 levels in the post AMI setting is supported by strong evidence of its damaging role on cells, such as its pro-apoptotic activity [START_REF] Pendergraft | Proteinase 3 sidesteps caspases and cleaves p21(Waf1/Cip1/Sdi1) to induce endothelial cell apoptosis[END_REF] and its pro-inflammatory effect, by releasing the active forms of both TNF , interleukin-1β, interleukin-18 [14-16] and the angiotensins [START_REF] Ramaha | Release and degradation of angiotensin I and angiotensin II from angiotensinogen by neutrophil serine proteinases[END_REF]. These cytokines may be especially relevant to development of heart failure post-AMI, which still occurs despite therapy with inhibitors of the renin-angiotensin system. The involvement of such serine proteases in the development of heart failure and their association with increased risk of adverse outcome post-AMI may support the search for new agents that could attenuate the damaging effects of these enzymes. For example, there is preliminary evidence that elafin, a serine protease inhibitor could reduce the vascular and myocardial damage in experimental models of cardiac transplantation [START_REF] Cowan | Elafin a serine elastase inhibitor, attenuates post-cardiac transplant coronary arteriopathy and reduces myocardial necrosis in rabbits afer heterotopic cardiac transplantation[END_REF].

The source of circulating PR3 may be partly from degranulating, activated neutrophils, being present in both azurophilic and secretory granules. Neutrophils are known to infiltrate myocardium after infarction, although their direct contribution to the development of heart failure to mortality after AMI has not been clarified. Alternatively, or additionally, PR3 may be secreted from endothelial cells [START_REF] Mayet | Human endothelial cells express proteinase 3, the target antigen of anticytoplasmic antibodies in Wegener's granulomatosis[END_REF] or from lung parenchymal cells [START_REF] Brockmann | Proteinase-3 as the major autoantigen of c-ANCA is strongly expressed in lung tissue of patients with Wegener's granulomatosis[END_REF]. Pneumocytes in normal lung have been demonstrated to express PR3 [START_REF] Brockmann | Proteinase-3 as the major autoantigen of c-ANCA is strongly expressed in lung tissue of patients with Wegener's granulomatosis[END_REF], although expression is greatly upregulated in Wegener's Granulomatosis. Active PR3 is rapidly inactivated in the plasma by SERPIN A1 and alpha2-macroglobulin [START_REF] Pham | Neutrophil serine proteases: specific regulators of inflammation[END_REF] the PR3-SERPIN A1 complex can therefore only be formed with an active protease and may therefore be a good measure of the amount of active enzyme that has been secreted. However, these plasma borne inhibitors may not have rapid access to extravascular sites where the enzyme could be released post-AMI. The levels of the PR3-SERPIN A1 complex that we have demonstrated in patients who have a poor outcome post-AMI are very similar to those described in patients with active vasculitis in Wegener's Granulomatosis (median 560 [range 110-3940] ng/ml) [START_REF] Ohlsson | Increased circulating levels of proteinase 3 in patients with anti-neutrophilic cytoplasmic autoantibodies-associated systemic vasculitis in remission[END_REF]. In normal subjects and those with inactive vasculitis, levels of about 350 (median [range 110-580] ng/ml) have been reported [START_REF] Ohlsson | Increased circulating levels of proteinase 3 in patients with anti-neutrophilic cytoplasmic autoantibodies-associated systemic vasculitis in remission[END_REF], which are lower than our values for our event-free post-AMI survivors. In this cohort of AMI patients, MPO did not provide independent prediction of death or heart failure, when models included NTproBNP and PR3-SERPIN A1. MPO is localised within neutrophil azurophilic granules alone, which are released with strong stimuli [START_REF] Pham | Neutrophil serine proteases: specific regulators of inflammation[END_REF], in contrast to the presence of easily mobilisable pool of PR3 in secretory granules [START_REF] Witko-Sarsat | Presence of proteinase 3 in secretory vesicles: evidence of a novel, highly mobilizable intracellular pool distinct from azurophil granules[END_REF]. In addition, previous studies with MPO have included patients with acute coronary syndromes or chest pain [START_REF] Brennan | Prognostic value of myeloperoxidase in patients with chest pain[END_REF][START_REF] Baldus | Myeloperoxidase serum levels predict risk in patients with acute coronary syndromes[END_REF] which may be a different spectrum from those with more myocardial damage. Indeed, Baldus et al [START_REF] Baldus | Myeloperoxidase serum levels predict risk in patients with acute coronary syndromes[END_REF] reported prognostic potential of MPO in those with low or medium Troponin T elevations, with poorer predictive potential in those with high Troponin T levels. Survival analysis revealed that those at highest risk were those with NTproBNP elevated above the median of 1023 pmol/L and PR3-SERPIN A1 above the median of 504.6 ng/ml. It is uncertain whether intensification of therapy or provision of timely interventional procedures could have altered clinical outcome, but this remains to be investigated.

Limitations of the study

This was a single centre study and the results need to be replicated in larger multicentre studies. There was a preponderance of ST elevation AMI, and the utility of PR3-SERPIN A1 in patients with other acute coronary syndromes should be investigated (eg in those with non-ST elevation AMI or chest pain). Larger studies may need to be performed to confirm the utility of PR3-SERPIN A1 in reclassification analyses.

Conclusion

This is the first report showing a potential role for the serine protease PR3 in determining mortality and incidence of heart failure following AMI, independent of established conventional risk factors and also the newly introduced biomarker NTproBNP. The involvement of PR3 in pathophysiology of Wegener's granulomatosis may extend to more common presentations of vascular disease. A multimarker approach with clinical risk scores and either or both of PR3-SERPIN A1 and NTproBNP may be useful for risk stratification in AMI patients, both markers providing complementary independent prediction, and the identification of such high risk patients may facilitate their subsequent management. A c c e p t e d M a n u s c r i p t

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Clinical

  Science Immediate Publication. Published on 14 Oct 2010 as manuscript CS20100366 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100366

  Clinical Science Immediate Publication. Published on 14 Oct 2010 as manuscript CS20100366 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100366A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

  4 [9.02-14057.2] pmol/L) or were readmitted with heart failure (3106.6 [2.4 -12282.9] pmol/L) compared to event free survivors (806.8 [0.3-28886] pmol/L; p<0.001 for both). NTproBNP levels were higher in females (1951.7 [22.9-15732] pmol/L) compared to males (871.6 [0.3-28886] pmol/ml; p<0.001) and correlated with age (r s = 0.46, p< 0.001), plasma glucose (r s = 0.15, p< 0.001), log creatinine (r s = 0.27, p< 0.001), Killip class (r s = 0.29, p< 0.001) and creatine kinase (r s =0.13, p< 0.001). Plasma MPO was significantly higher in patients who died (36.7 [6.44-132.1] ng/ml, P<0.035 using Bonferroni's correction) compared to event free survivors (24.6 [0.3-405.2] ng/ml). In contrast those who were readmitted with heart failure(27.6 [6.5-210.8] ng/ml) or with recurrent AMI (23.9[5.3-189.9] ng/ml) had similar values of MPO to event free survivors. MPO levels were higher in females (30.3 [0.3-405.2] ng/ml) compared to males (23.9 [3.6-215.1] ng/ml; p<0.001) and in patients with diabetes (30.4[5.1-238.7] ng/ml) compared to non-diabetic patients (24.0 [0.3-405.2] ng/ml; p<0.001) and were correlated with age (r s = 0.12, p< 0.001), plasma glucose (r s = 0.10 p< 0.009, log creatinine (r s = 0.11, p< 0.001), and Killip class (r s = 0.11, p< 0.002).

Clinical

  Science Immediate Publication. Published on 14 Oct 2010 as manuscript CS20100366 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100366A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Clinical

  Science Immediate Publication. Published on 14 Oct 2010 as manuscript CS20100366 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100366 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. Secondary Endpoints: PR3-SERPIN A1 and NTproBNP as predictors of myocardial infarction Compared to survivors with no endpoints, patients who were readmitted with further AMI during follow up had similar NTproBNP (median [range] 1480.7[2.6-10645.9] vs. 806.8[0.3-28886] pmol/L; p=NS), PR3-SERPIN A1 levels (median [range] 482.5[168.9-1753.9] vs. 486.9[29.3-3118.2] ng/ml; p=NS) and MPO levels (23.9 [5.3-189.9] vs. 24.6 [0.3-405.2] ng/ml; p=NS).

Clinical

  Science Immediate Publication. Published on 14 Oct 2010 as manuscript CS20100366 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100366A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Figure 1 :

 1 Figure 1: Cumulative incidence plots showing time to adverse events (death or heart failure) in patients stratified by PR3-SERPIN A1.

Figure 2 :

 2 Figure 2: Cumulative incidence plots showing time to adverse events (death or heart failure) in patients stratified according to whether both PR3-SERPIN A1 and NTproBNP are below their respective median values, either marker being elevated, or both markers elevated above median.

  Clinical Science Immediate Publication. Published on 14 Oct 2010 as manuscript CS20100366 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100366

Table 1

 1 Characteristics of patients in the study. Values are means (SD) or numbers (%)

		AMI Patients
	Number (male)	900 (700)
	Age (in years)	64.6 12.4
	Previous Medical History
	Myocardial infarction 165 (18.3)
	Angina Pectoris	195 (21.7)
	Hypertension	391 (43.4)
	Diabetes mellitus	192 (21.3)
	Hypercholesterolaemia 221 (24.6)
	Current/Ex-Smokers	560 (62.2)
	ST-elevation AMI	777 (86.3)
	Thrombolytic	529 (58.8)
	Territory of Infarct
	Anterior	369 (41.0)
	Inferior	373 (41.4)
	Other/undetermined	158 (17.6)
	Killip Class on Admission
	I	457 (50.7)
	II	357 (39.7)
	III	77 (8.6)
	IV	9 (1.0)
	Peak CK (IU/L)	1157 1263
	Creatinine (µmol/L)	103.9 35.4
	Medication
	Aspirin	794 (88.2)
	ACE inhibitors/	683 (75.9)
	Angiotensin receptor blockers
	Beta blockers	721 (80.1)
	Statins	701 (77.9)

Table 2

 2 Binary Logistic regression model for prediction of death and heart failure

	Variable	Odds Ratio	95% CI	p value
	Age	1.04	1.02-1.07	0.001
	Gender	.57	0.35-0.93	0.024
	Past history AMI	2.28	1.43-3.74	0.001
	Killip Class >1	1.66	1.05-2.63	0.032
	Log Creatinine	18.55	3.02-113.9	0.001
	Log NTproBNP	2.79	1.79-4.37	0.001
	Log PR3-SERPIN A1	6.42	2.25-18.3	0.001

Table 3

 3 Multivariate Cox proportional hazards regression model of significant predictors of death or heart failure

	Variable	Hazard Ratio	95% CI	p value
	Age	1.04	1.03-1.06	0.001
	Past history AMI	1.52	1.06-2.17	0.023
	Killip Class >1	1.60	1.08-2.37	0.02
	Log Creatinine	4.75	1.39-16.19	0.013
	Log NTproBNP	2.51	1.70-3.71	0.001
	Log PR3-SERPIN A1	3.80	1.78-8.14	0.001

Table 4

 4 Reclassification analysis for death or heart failure using GRACE score, with the addition of NTproBNP and/or PR3-SERPIN A1 GRACE score vs GRACE score with NTproBNP Clinical Science Immediate Publication. Published on 14 Oct 2010 as manuscript CS20100366 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100366

		Reclassification	% Correctly	p value
				Reclassified
		Increased %	Decreased %
	Patients without endpoints	8.9	16.3	7.4	0.0002
	Patients with endpoints	15.3	7.0	8.3
	GRACE score vs GRACE score with PR3-SERPIN A1
	Patients without endpoints	10.3	11.4	1.1	0.02
	Patients with endpoints	9.1	2.3	6.8
	GRACE score with NTproBNP vs GRACE score with NTproBNP and PR3-SERPIN A1
	Patients without endpoints	8.6	7.9	-0.7	0.14
	Patients with endpoints	7.6	2.3	5.3

© 2010 The Authors Journal compilation © 2010 Portland Press Limited

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.© 2010 The Authors Journal compilation © 2010 Portland Press Limited

Acknowledgements

This study is part of the research portfolio supported by the Leicester NIHR Biomedical Research Unit in Cardiovascular Disease. SQK was supported by a BHF Junior Research Fellowship FS/03/028/15486


María Elena Lima-Cabello

Victoria García-Mediavilla

María E Miquilena-Colina

Javier Vargas-Castrillón

Tamara Lozano-Rodríguez

Miguel Fernández-Bermejo

José L Olcoz

Javier González-Gallego

Carmelo García-Monzón

Sonia Sánchez-Campos

Victoria García

María Elena Lima-Cabello1

Victoria García-Mediavilla1

María E Miquilena- Colina2

Javier Vargas-Castrillón2

Tamara Lozano-Rodríguez2

Miguel Fernández-Bermejo3

José Luis Olcoz4

Javier González-Gallego1

Carmelo García-Monzón2

Sonia Sánchez-Campos1

Enhanced expression of pro-inflammatory mediators and liver X receptor-regulated lipogenic genes in

Keywords: Liver X receptor, nonalcoholic fatty liver disease, chronic HCV infection, lipogenesis, inflammation NAFLD, Nonalcoholic fatty liver disease, NASH, nonalcoholic steatohepatitis, SREBP, sterol regulatory element binding protein, TNF, tumor necrosis factor, IL, interleukin, HCV, hepatitis C virus, LXR, liver X receptor, iNOS, inducible nitric oxide synthase, COX, cyclooxygenase, OPN, osteopontin, ALT, alanine aminotransferase, γ-GT, gamma-glutamyltrasferase, NAS, nonalcoholic steatosis, NL, normal liver, PPAR-γ, peroxisome proliferator-activated receptor gamma, FAS, fatty acid synthase, CYP2E1, cytochrome P450 2E1, SOCS, suppressors

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Nonalcoholic fatty liver disease (NAFLD) is one of the most frequent causes of abnormal liver function and correlates with central adiposity, obesity, insulin resistance, the metabolic syndrome and type 2 diabetes mellitus [START_REF] Mccullough | The clinical features, diagnosis and natural history of nonalcoholic fatty liver disease[END_REF]. The pathological spectrum of NAFLD ranges from fatty liver (steatosis) to nonalcoholic steatohepatitis (NASH), advanced fibrosis, cirrhosis, and even hepatocellular carcinoma [START_REF] Angulo | Nonalcoholic fatty liver disease[END_REF]. Accumulation of excess triglyceride in hepatocytes is necessary for the development of NAFLD. Fat either is delivered to hepatocytes in the form of free fatty acids bound to albumin or is synthesized de novo within hepatocytes [START_REF] Neuschwander-Tetri | Nonalcoholic steatohepatitis and the metabolic syndrome[END_REF]. In this regard, several lines of evidence indicate that hepatic activation of sterol regulatory element binding protein-1c (SREBP-1c) together with other lipogenic or adipogenic transcription factors is crucial for the development of fatty liver [START_REF] Joseph | Direct and indirect mechanisms for regulation of fatty acid synthase gene expression by liver X receptors[END_REF][START_REF] Yoshikawa | Cross-talk between peroxisome proliferators-activated receptor (PPAR) alpha and liver X receptor (LXR) in nutritional regulation of fatty acid metabolism. I. PPARs suppress sterol regulatory element binding protein-1c promoter through inhibition of LXR signalling[END_REF]. While the mechanisms involved in the transition from bland steatosis to NASH have not been fully established so far, there is increasing evidence on the central role of nontriglyceride lipotoxicity in the pathogenesis of NASH [START_REF] Neuschwander-Tetri | Hepatic lipotoxicity and the pathogenesis of nonalcoholic steatohepatitis: the central role of nontriglyceride fatty acid metabolites[END_REF]. In this hypothesis, the accumulation of triglyceride in the form of lipid droplets within the liver is not needed for the development of NASH and, in fact, it may be protective whereas toxic metabolites derived from free fatty acids could lead to NASH by inducing endoplasmic reticulum stress, inflammation and liver cell death by apoptosis [START_REF] Day | From fat to inflammation[END_REF][START_REF] Neuschwander-Tetri | Nontriglyceride hepatic lipotoxicity: the new paradigm for the pathogenesis of NASH[END_REF].

Hepatic steatosis is a common histologic feature in chronic hepatitis C virus (HCV) infection and has been reported to be associated with fibrosis [START_REF] Lonardo | Hepatitis C and steatosis: a reappraisal[END_REF]. HCV has been shown to alter host cell cholesterol/lipid metabolism and induce hepatic steatosis [10]. Furthermore, HCV proteins associate with mitochondria and endoplasmic reticulum and promote oxidative stress, increasing gene expression of oxidative stress-mediated inflammatory genes [START_REF] Gochee | Steatosis in chronic hepatitis C: association with increased messenger RNA expression of collagen I, tumor necrosis factor-alpha and cytochrome P450 2E1[END_REF][START_REF] Sheikh | Hepatitis C virus infection: molecular pathways to metabolic syndrome[END_REF][START_REF] García-Mediavilla | Differential contribution of hepatitis C virus NS5A and core proteins to the induction of oxidative and nitrosative stress in human hepatocyte-derived cells[END_REF].

Liver X receptors (LXRs) are members of the nuclear hormone receptor superfamily that work as fatty acid-activated transcription factors [START_REF] Repa | Regulation of mouse sterol regulatory element-binding protein-1c gene (SREBP-1c) by oxysterol receptors, LXRalpha and LXRbeta[END_REF]. Considerable evidence has emerged indicating that, besides to induce SREBP-1c-mediated hepatic fatty acid biosynthesis [START_REF] Joseph | Direct and indirect mechanisms for regulation of fatty acid synthase gene expression by liver X receptors[END_REF][START_REF] Repa | Regulation of mouse sterol regulatory element-binding protein-1c gene (SREBP-1c) by oxysterol receptors, LXRalpha and LXRbeta[END_REF], LXRs repress in macrophages a set of inflammatory genes as IL-6, inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2 and osteopontin (OPN) [START_REF] Joseph | Reciprocal regulation of inflammation and lipid metabolism by liver X receptors[END_REF][START_REF] Ogawa | Liver X receptor agonists inhibit cytokine-induced osteopontin expression in macrophages through interference with activator protein-1 signaling pathways[END_REF]. The latter is considered as a pro-inflammatory cytokine strongly associated with the early stages of development of NASH in animal models [START_REF] Sahai | Upregulation of osteopontin expression is involved in the development of non-alcoholic steatohepatitis in a dietary murine model[END_REF]. Albeit hepatic mRNA and protein levels of iNOS and COX-2 are increased in HCV patients [18,[START_REF] Núñez | Increased intrahepatic cyclooxygenase 2, matrix metalloproteinase 2, and matrix metalloproteinase 9 expression is associated with progressive liver disease in chronic hepatitis C virus infection: role of viral core and NS5A proteins[END_REF], little is known, however, on the role of LXRs in the development of hepatic steatosis and inflammation in chronic HCV infection. Based on these data, it is conceivable that LXR may be a key regulator of hepatic lipogenesis and inflammation in NAFLD and HCV patients. In order to shed light on this hypothesis, we carried out this study aimed to determine hepatic mRNA and protein levels of LXRα and its related lipogenic and inflammatory targets in patients with NAFLD, chronic HCV infection, and histologically normal liver (NL).

PATIENTS AND METHODS

Patients. This study comprised 87 nondiabetic patients with a clinical diagnosis of either NAFLD or chronic HCV infection who underwent liver biopsy with diagnostic purposes. Inclusion criteria for NAFLD patients were based on the absence of alcohol intake, the presence of biopsy-proven steatosis with/without necroinflammation and/or fibrosis, and a negative anti-HCV serum test. Patients with chronic HCV infection were included if they had serum anti-HCV and HCV RNA, persistently abnormal serum alanine aminotransferase (ALT) levels, liver histology findings compatible with chronic hepatitis, and a negative history of alcohol consumption. In order to avoid bias depending of HCV genotype, all HCV patients included were infected by genotype 1. In addition, none of HCV patients studied had ever received antiviral therapy. Patients with other potential causes of chronic liver disease or those receiving drug treatment for any chronic medical disorder were excluded.

We further studied 22 patients with asymptomatic cholelithiasis to whom a liver biopsy was taken, during programmed laparoscopic cholecystectomy, as part of a study protocol and after signing a specific informed consent. All these subjects were otherwise healthy, and had histologically normal liver, normal fasting glucose, cholesterol and triglycerides, normal AST and ALT, and no evidence of viral infections (hepatitis B virus, HCV and human immunodeficiency virus). In addition, none of these individuals drank alcohol.

The study was performed in agreement with the Declaration of Helsinki and with local and national laws. The Institution´s Human Ethics Committee approved the study procedures, and written informed consent was obtained from all patients before inclusion in the study. Clinical and laboratory assessment. After a 12-hour overnight fast, clinical and anthropometric data of each patient were collected at the time of liver biopsy as well as venous blood samples to test hepatic enzymes and metabolic parameters by using a conventional automated analyzer. Serum insulin was determined by a chemiluminescent microparticle immunoassay (ARCHITECT insulin; Abbot Park, IL). Insulin resistance was calculated by the homeostasis model assessment (HOMA-IR) [START_REF] Matthews | Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man[END_REF].

Antibodies against HCV and HIV as well as hepatitis B surface antigen were tested by immunoenzymatic assays (Murex, Dartford, UK). HCV RNA levels were quantified by real-time PCR (COBAS AmpliPrep/COBAS TaqMan HCV test, Roche Diagnostics, Branchburg, NJ). HCV genotyping was performed using a second generation line-probe hybridization assay (HCV genotype 2.0 assay (LiPA), Roche Diagnostics, Branchburg, NJ). Liver tissue studies. Liver biopsies were divided in at least two pieces of a minimum length of 15 mm each. One of them was fixed in buffered formalin and used for routine histological staining whereas the remaining pieces were immediately snap-frozen and stored in liquid nitrogen. Histopathology assessment: Paraffin-embedded liver biopsy sections were evaluated under code by a single hepatopathologist blinded to the clinical data. Overall, steatosis was assessed as outlined by Brunt et al [START_REF] Brunt | Nonalcoholic steatohepatitis: a proposal for grading and staging the histological lesions[END_REF] and the Kleiner´s histological scoring system [START_REF] Kleiner | Nonalcoholic Steatohepatitis Clinical Research Network. Desing and validation of a histological scoring system for nonalcoholic fatty liver disease[END_REF] was used to assess the grade of necroinflammation and the stage of fibrosis in NAFLD patients who were classified into two groups: those with simple steatosis (NAS) and those with definite steatohepatitis (NASH). In the HCV group, the grade of necroinflammation and the stage of fibrosis were scored as proposed by Scheuer [START_REF] Scheuer | Classification of chronic viral hepatitis: a need for reassessment[END_REF]. HCV patients were categorized into two groups, depending of the presence of steatosis. Histologic photomicrographs were acquired by using a high-resolution digital videocamera (Nikon DXM 1200, Tokyo, Japan) connected to a light microscope (Nikon Eclipse E400, Tokyo, Japan) equipped with a planApocromatic 20x objective. Quantitative real-time PCR. Total RNA of liver biopsy samples from all patients was extracted by using a Trizol reagent (Life Technologies, Carlsbad, CA). First-standard cDNA was synthesized using High-Capacity cDNA Archive Kit (Applied Biosystems, Weiterstadt, Germany). cDNA was amplified using multiplex real-time PCR reactions on a StepOne Plus (Applied Biosystems). TaqMan primers and probes were derived from the commercially available TaqMan® Gene Expression Assays (Applied Biosystems) (Table 1). Relative changes in gene expression levels were determined using the 2 -∆∆Ct method. The cycle number at which the transcripts were detectable (CT) was normalized to the cycle number of GAPDH detection, referred to as ∆CT. Western blot. Protein extraction and western blotting were performed as described [START_REF] García-Mediavilla | Differential contribution of hepatitis C virus NS5A and core proteins to the induction of oxidative and nitrosative stress in human hepatocyte-derived cells[END_REF] with modifications. Briefly, liver biopsy samples from all patients of each group were homogeneized in a medium containing: 0.25 mM sucrose, 1 mM EDTA, 10 mM Tris and 1% protease inhibitor cocktail (Roche, Mannheim, Germany). For western blot analysis, total protein were boiled in Laemmli sample buffer and equal amounts of protein (30-50 µg) were separated by SDS-polyacrylamide gel electrophoresis (12% acrylamide) and transferred to PVDF membranes (Millipore, Massachusetts, United States). Nonspecific binding was blocked by preincubation of the membranes in phosphate-buffered saline containing 5% skimmed milk for 1 h. Membranes were incubated overnight at 4°C with primary antibodies as rabbit polyclonal antibodies against LXRα (Abcam, Cambridge, UK), SREBP-1c (Abcam), PPAR-γ (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), TNF-α (Abcam), and iNOS (BIOMOL International L.P., Plymouth Meeting, PA) or mouse monoclonal antibody against OPN (Abcam). Bound primary antibody was detected with HRP-conjugated anti-rabbit or anti-mouse antibodies (DAKO, Glostrup, Denmark) and blots were developed using an enhanced chemiluminescence detection system (ECL kit, Amersham Pharmacia, Uppsala, Sweden). The density of the specific bands was quantified with an imaging densitometer (Scion Image, Maryland, MA). Statistical analysis. Categorical variables are presented as frequency and percentage. Continuous variables are shown as mean ± standard deviation. The baseline characteristics of the patients studied were compared by the Pearson χ 2 test for categorical variables and the unpaired t test or Mann-Whitney U test for continuous variables. Data from qPCR and Western blotting were compared by using the Kruskal-Wallis ANOVA test. All statistical analysis were performed using SPSS version 15.0 (SPSS, Cary, NC) software with 2-sided tests, with a p value of < 0.05 considered as statistically significant.

RESULTS

Patient characteristics.

Characteristics of the study population are detailed in Tables 2 and3. To highlight, all patient groups were well matched in terms of age and sex distribution. NAS patients had significantly higher serum levels of triglycerides (p = 0.008) and γ-GT (p = 0.037) than NL subjects. In NASH patients, the serum insulin levels and the mean HOMA-IR score were significantly higher than in NL individuals (p = 0.017 and p = 0.004, respectively), being the higher serum levels of ALT the only significant difference (p = 0.009) with respect to NAS patients. No significant differences were observed between HCV patient groups.

At liver biopsy, the degree of steatosis was similar in NAS and NASH patients (Fig. 1, panels A andB). On the other hand, comparing HCV patients with or without steatosis (Fig. 1, panels C and D), no significant differences were found in the grade of necroinflammation and in the stage of fibrosis.

Hepatic expression of LXRα and related lipogenic genes is enhanced in NAFLD patients and in HCV patients with steatosis.

When compared with NL, hepatic mRNA levels of LXRα and PPAR-γ were significantly increased in NAS (+114% and +131%, respectively) and NASH (+131% and +120%, respectively) (Fig. 2, panel A and Fig. 3,panel A). The significant induction in LXRα and PPAR-γ was also shown in HCV patients, being higher in those with steatosis (HCV: +41%, HCV+NAS: +126%; and HCV. +44% and HCV+NAS: +132%, respectively, vs NL) (Fig. 2, panel A and Fig. 3, panel A). Hepatic mRNA levels of SREBP-1c were significantly higher in NAS (+81%) and NASH (+124%) than in NL (Fig. 3, panel B). Regarding HCV patients, hepatic mRNA levels of SREBP-1c were significantly increased with respect to NL only in those with steatosis (+165%) (Fig. 3, panel B). We also found that hepatic mRNA levels of SREBP-2 were significantly higher in NAS (+190%) and NASH (+80%) than in NL (Fig. 3,panel C). Only HCV patients with steatosis had hepatic mRNA levels of SREBP-2 significantly more elevated than NL (+130%) (Fig. 3,panel C). Hepatic gene expression of FAS showed a similar pattern to that observed for LXRα and PPAR-γ (Fig. 3, panel D).

Hepatic content of LXRα, PPAR-γ and SREBP-1c proteins was significantly increased in NAS (+112%, +86%, +126%; respectively) and NASH (+166%, +89%, +176%; respectively) than in NL (Fig. 2, panel B and Fig. 4). Noteworthy, protein amounts of LXRα and SREBP-1c were significantly higher in liver biopsies from patients with NASH than in those with NAS (Fig. 2, panel B and Fig. 4). In addition, a significant increase in LXRα, PPAR-γ and SREBP-1c protein content was shown in HCV patients (+48%, +46%, +43%, respectively, vs NL), being the highest LXRα, PPAR-γ and SREBP-1c levels observed in HCV patients who had steatosis (+155%, +276%,+191%, respectively, vs NL) (Fig. 2, panel B and Fig. 4).

Hepatic expression of oxidative stress and inflammatory genes is upregulated in NAFLD and HCV patients.

Overexpression of cytochrome P450 2E1 (CYP2E1) leads to oxidative stress [START_REF] Leclercq | CYP2E1 and CYP4A as microsomal catalysts of lipid peroxides in murine nonalcoholic steatohepatitis[END_REF]. Thus, we wanted to determine hepatic CYP2E1 gene expression in our study population. We found that CYP2E1 mRNA levels were significantly increased in the liver of patients with NAS (+74%) and NASH (+72%) with respect to NL (Fig. 5, panel A). To note, only HCV patients with steatosis had hepatic CYP2E1 mRNA levels significantly higher than NL (+105%) (Fig. 5,panel A).

We further analyzed hepatic mRNA levels of LXR-related inflammatory genes such as TNF-α, IL-6, OPN, iNOS and COX-2. Intrahepatic OPN, TNF-α, and IL-6 mRNA levels were significantly increased in NAS (+271%, +123% and +300%, respectively) and NASH (+116%, +105% and +188%, respectively) as compared with NL (Fig. 5, panels B, C, and D). Interestingly, OPN and IL-6 mRNA levels were significantly decreased in NASH (-57%, and -37%, respectively) with respect to NAS (Fig. 5, panels B and D). However, no differences in the hepatic OPN and IL-6 mRNA levels between HCV patients with or without steatosis were observed (Fig. 5, panels B, and D). In contrast, TNF-α mRNA levels were significantly higher in HCV patients with steatosis (+25%) than in those without steatosis (Fig. 5, panel C). In addition, we found that hepatic suppressors of cytokine signaling (SOCS)-3 mRNA levels were significantly increased in NAS (+92%) and NASH (+107%) with respect to NL, being the highest SOCS-3 mRNA levels observed in HCV patients regardless of the presence of steatosis (Fig. 5, panel E). We also found hepatic iNOS and COX-2 mRNA levels were significantly enhanced in NAS (+87% and +105%, respectively) and NASH (+102% and +130%, respectively) as well as in HCV patients without steatosis (+221% and +524%, respectively) and with steatosis (+250% and +329%, respectively) compared with NL (Fig. 6, panels A andB).

On the other hand, a statistically significant increase in the hepatic abundance of OPN, TNF-α and iNOS proteins was observed in NAS (+137%, +172%, and +98%, respectively) and NASH (+105%, +98% and +104%, respectively) with respect to NL (Fig. 7). Noteworthy, the hepatic content of OPN and TNF-α was significantly lower in NASH patients than in those with NAS (Fig. 7). We also found a significant increase of OPN and iNOS proteins in HCV patients without steatosis (+309% and +137%, respectively) and with steatosis (+317% and +189%, respectively, vs NL) Interestingly, in HCV patients, TNF-α protein content was highest when steatosis was present (HCV: +229% and HCV+NAS: +287%, vs NL) (Fig. 7).

DISCUSSION

There is experimental evidence that LXRα induces the expression of lipogenic genes, such as PPARγ and SREBP-1c, and downregulates the expression of inflammatory genes, such as TNFα, IL-6, and osteopontin [START_REF] Repa | Regulation of mouse sterol regulatory element-binding protein-1c gene (SREBP-1c) by oxysterol receptors, LXRalpha and LXRbeta[END_REF][START_REF] Joseph | Reciprocal regulation of inflammation and lipid metabolism by liver X receptors[END_REF]. This study demonstrates that LXRα and its related lipogenic and inflammatory gene targets are upregulated in liver biopsies from NALFD and HCV patients with steatosis. Given the key role of LXR signalling pathways in the cross-talk between lipogenesis and inflammation [START_REF] Joseph | Reciprocal regulation of inflammation and lipid metabolism by liver X receptors[END_REF], we propose a direct contribution of LXRα in the development of NAFLD as well as in HCV-associated steatosis.

LXRs belong to the nuclear hormone receptor family and are DNA-binding transcriptional regulators. LXRs form heterodimers with the retinoid X receptor (RXR)α and bind to target DNA sequences known as the LXR response element (LXRE) [START_REF] Willy | LXR, a nuclear receptor that defines a distinct retinoid response pathway[END_REF]. There are two isoforms termed LXRα and LXRβ, both expressed in the liver among other locations [START_REF] Repa | The role of orphan nuclear receptors in the regulation of cholesterol homeostasis[END_REF]. In response to oxysterols, LXRs activate genes involved in reverse cholesterol transport and hepatic cholesterol metabolism [START_REF] Repa | The role of orphan nuclear receptors in the regulation of cholesterol homeostasis[END_REF]. Hepatic lipogenesis is generally induced by nuclear receptors such as PPAR-γ and LXRα [START_REF] Yoshikawa | Cross-talk between peroxisome proliferators-activated receptor (PPAR) alpha and liver X receptor (LXR) in nutritional regulation of fatty acid metabolism. I. PPARs suppress sterol regulatory element binding protein-1c promoter through inhibition of LXR signalling[END_REF][START_REF] Repa | Regulation of mouse sterol regulatory element-binding protein-1c gene (SREBP-1c) by oxysterol receptors, LXRalpha and LXRbeta[END_REF][START_REF] Larter | Insulin resistance, adiponectin, cytokines and NASH: Which is the best target to treat?[END_REF]. These transcription factors increase the synthesis of fatty acids by either upregulating SREBP-1c or directly binding to the promoters of some lipogenic genes, including FAS [START_REF] Joseph | Direct and indirect mechanisms for regulation of fatty acid synthase gene expression by liver X receptors[END_REF][START_REF] Repa | Regulation of mouse sterol regulatory element-binding protein-1c gene (SREBP-1c) by oxysterol receptors, LXRalpha and LXRbeta[END_REF][START_REF] Schadinger | PPARgamma2 regulates lipogenesis and lipid accumulation in steatotic hepatocytes[END_REF]. LXRα may play a role in enhancing lipogenesis in the liver of NAFLD patients, as previously described [START_REF] Higuchi | Liver X receptor in cooperation with SREBP-1c is a major lipid synthesis regulator in non-alcoholic fatty liver disease[END_REF][START_REF] Nakamuta | Impact of cholesterol metabolism and the LXRalpha-SREBP-1c pathway on non-alcoholic fatty liver disease[END_REF]. In the current study, hepatic LXRα, PPAR-γ, SREBP-1c, SREBP-2 and FAS gene expression was significantly increased in NAFLD patients with respect to NL and, interestingly, the hepatic content of LXRα protein was significantly higher in NASH than in NAS. These findings suggest a steatogenic role for LXRα and its target lipogenic genes PPAR-γ, SREBP-1c, SREBP-2 and FAS. Different factors could induce hepatocyte LXR expression, such as saturated and unsaturated fatty acids, PPAR-γ, LXR agonists and lipid peroxidation products (by stimulating PPAR-γ) [START_REF] Wójcicka | Liver X receptors (LXRs). Part I: structure, function, regulation of activity, and role in lipid metabolism[END_REF][START_REF] Kim | Interrelationship between liver X receptor alpha, sterol regulatory element-binding protein-1c, peroxisome proliferator-activated receptor gamma, and small heterodimer partner in the transcriptional regulation of glucokinase gene expression in liver[END_REF]. In turn, LXR increases the transcriptional activity and gene expression of PPAR-γ through the LXRE contained in its promoter region [START_REF] Kim | Interrelationship between liver X receptor alpha, sterol regulatory element-binding protein-1c, peroxisome proliferator-activated receptor gamma, and small heterodimer partner in the transcriptional regulation of glucokinase gene expression in liver[END_REF]. In addition, LXRα expression is controlled by an auto-regulatory mechanism, since the human LXRα gene promoter contains three functional LXRE [START_REF] Willy | LXR, a nuclear receptor that defines a distinct retinoid response pathway[END_REF]. This mechanism may be responsible for the strong upregulation of LXRα in lipidloaded hepatocytes.

A novel finding of our study is that hepatic mRNA and protein levels of LXRα and its downstream lipogenic genes were abnormally increased in HCV patients who had steatosis. Mechanisms involved in HCV-induced steatosis seem to be mediated by HCV proteins, whose expression is associated with changes in lipogenic gene expression and/or the activity of lipogenic proteins and effects on mitochondrial oxidative function [10,[START_REF] Kim | Interrelationship between liver X receptor alpha, sterol regulatory element-binding protein-1c, peroxisome proliferator-activated receptor gamma, and small heterodimer partner in the transcriptional regulation of glucokinase gene expression in liver[END_REF]. Thus, HCV core protein indirectly enhances the transcriptional activity of SREBP-1c, increasing the binding of LXRα/RXRα to LXRE as described in an in vivo model [START_REF] Moriishi | Critical role of PA28gamma in hepatitis C virus-associated steatogenesis and hepatocarcinogenesis[END_REF]. Accordingly, HCV core protein may play a crucial role in the development of hepatic steatosis by activating the LXRα signalling pathway in HCV-infected hepatocytes. In addition, HCV NS2 protein is able to induce the transcription of SREBP-1c and FAS, and this could be a contributing factor for HCV-associated steatosis [START_REF] Oem | Activation of sterol regulatory element-binding protein 1c and fatty acid synthase transcription by hepatitis C virus non-structural protein 2[END_REF].

When an excess of fatty acids occurs in hepatocytes, CYP2E1 gene expression is induced initiating the process of lipid peroxidation [START_REF] Leclercq | CYP2E1 and CYP4A as microsomal catalysts of lipid peroxides in murine nonalcoholic steatohepatitis[END_REF]. In our study, we found that hepatic mRNA levels of CYP2E1 were increased in NAFLD patients, as previously indicated [START_REF] Mitsuyoshi | Analysis of hepatic genes involved in the metabolism of fatty acids and iron in non-alcoholic fatty liver disease[END_REF]. Interestingly, CYP2E1 gene expression is also enhanced in HCV patients who had steatosis. The latter is in agreement with previous observations by A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Gochee et al. [START_REF] Gochee | Steatosis in chronic hepatitis C: association with increased messenger RNA expression of collagen I, tumor necrosis factor-alpha and cytochrome P450 2E1[END_REF], supporting the notion that hepatic CYP2E1 upregulation is not a direct effect of HCV.

In fatty liver, distinct mechanisms such as oxidative stress, endoplasmic reticulum stress and hypoadiponectinemia are able to activate a set of inflammatory pathways including secretion of inflammatory cytokines, such as TNF-α and IL-6 [START_REF] Hui | Beyond insulin resistance in NASH: TNF-alpha or adiponectin?[END_REF]. In particular, TNF-α promotes triglyceride accumulation and hepatic lipotoxicity, at least in part, by inducing hepatocyte lysosomal destabilisation [37]. Our current results show an increased hepatic TNF-α, IL-6 and SOCS-3 gene expression in patients with NAFLD, while TNF-α protein and IL-6 mRNA levels were significantly lower in NASH than in NAS patients. These findings argue against a direct role of TNF-α and IL-6 as pathogenic mediators in NASH. However, many of the biological effects of TNF-α are mediated by IL-6, and experimental evidence exists showing that IL-6 induces hepatic SOCS-3 expression [START_REF] Yang | Interleukin-6 plays a crucial role in the hepatic expression of SOCS3 during acute inflammatory processes in vivo[END_REF]. Since SOCS-3 promotes SREBP-1c expression and subsequent development of hepatic steatosis in mice [START_REF] Ueki | Central role of suppressors of cytokine signaling proteins in hepatic steatosis, insulin resistance, and the metabolic syndrome in the mouse[END_REF], our findings strongly suggest a key role for SOCS-3 in the development of steatosis in NAFLD patients. On the other hand, we also found an enhanced hepatic TNF-α and IL-6 gene expression in HCV patients, being the highest TNF-α mRNA and protein levels observed in those HCV patients who had steatosis. The latter is in agreement with previous observations [START_REF] Gochee | Steatosis in chronic hepatitis C: association with increased messenger RNA expression of collagen I, tumor necrosis factor-alpha and cytochrome P450 2E1[END_REF], suggesting that TNF-α may play an important role in the development of HCV-associated steatosis. Additionally, SOCS-3 gene expression is increased in HCV patients regardless of fat accumulation as previously described in genotype 1 non-responders subjects [START_REF] Persico | Elevated expression and polymorphisms of SOCS3 influence patient response to antiviral therapy in chronic hepatitis C[END_REF].

Upregulation of OPN gene expression has been implicated early in the development of NASH in a murine model [START_REF] Sahai | Upregulation of osteopontin expression is involved in the development of non-alcoholic steatohepatitis in a dietary murine model[END_REF]. In the present study we found that OPN gene expression is markedly enhanced in NAS patients while significantly decreased in those with NASH, in a similar extent to that observed for TNF-α protein and IL-6 mRNA levels. Noteworthy, the highest LXRα protein levels were observed in the same NASH patients. As previously reported, this transcription factor might modulate hepatic inflammation by inhibiting TNF-α, IL-6 and OPN gene expression in Kupffer cells [START_REF] Joseph | Reciprocal regulation of inflammation and lipid metabolism by liver X receptors[END_REF][START_REF] Ogawa | Liver X receptor agonists inhibit cytokine-induced osteopontin expression in macrophages through interference with activator protein-1 signaling pathways[END_REF][START_REF] Wang | Activation of the liver X receptor protects against hepatic injury in endotoxemia by suppressing Kupffer cell activation[END_REF]. The mechanism underlying the repression of inflammatory genes by LXR is poorly understood. LXREs have not been identified in the proximal promoters of the repressed genes [START_REF] Joseph | Reciprocal regulation of inflammation and lipid metabolism by liver X receptors[END_REF][START_REF] Ogawa | Liver X receptor agonists inhibit cytokine-induced osteopontin expression in macrophages through interference with activator protein-1 signaling pathways[END_REF], indicating an indirect mechanism through the regulation of other transcription factors supporting gene transcription. Elucidating whether hepatic upregulation of LXRα occurs in hepatocytes or in Kupffer cells may help to understand the inhibitory action of this transcription factor on inflammatory gene expression in NAFLD patients.

On the other hand, our results also show that hepatic OPN gene expression is markedly increased in HCV patients regardless of the presence of steatosis, as previously indicated regarding SOCS-3 and IL-6 mRNA levels, favouring the hypothesis that inflammatory gene upregulation might be a direct effect of HCV itself. In our study, the highest LXRα protein levels observed in HCV-related steatosis is not accompanied by a repression of OPN, IL-6 and SOCS-3 gene expression. Thus, there is no compelling evidence for a role of LXRα in modulating inflammation in HCV.

iNOS and COX-2 play an important role in development and progression of liver injury in NASH patients [START_REF] Giannitrapani | Cyclooxygenase-2 expression in chronic liver diseases and hepatocellular carcinoma: an immunohistochemical study[END_REF][START_REF] García-Monzón | Characterization of pathogenic and prognostic factors of non-alcoholic steatohepatitis associated with obesity[END_REF]. Our study results confirm these observations and further show that hepatic iNOS and COX-2 gene expression is similarly increased in NAS and NASH, suggesting that hepatic iNOS and COX-2 upregulation is an early event remaining unchanged in the course of NAFLD. In addition, we also found that hepatic iNOS gene expression is increased in HCV patients, being the highest iNOS protein content observed in HCV patients who had steatosis. These findings are in agreement with experimental evidence from our group indicating that HCV NS5A and core proteins are able to upregulate iNOS gene expression [START_REF] García-Mediavilla | Differential contribution of hepatitis C virus NS5A and core proteins to the induction of oxidative and nitrosative stress in human hepatocyte-derived cells[END_REF]. An interesting finding of the present study is that hepatic COX-2 mRNA levels were significantly lower in HCV patients with steatosis than in those without fatty liver. There is experimental evidence that HCV core protein can induce COX-2 expression in human hepatocytes [START_REF] Núñez | Increased intrahepatic cyclooxygenase 2, matrix metalloproteinase 2, and matrix metalloproteinase 9 expression is associated with progressive liver disease in chronic hepatitis C virus infection: role of viral core and NS5A proteins[END_REF], but whether fat accumulation may modulate COX-2 expression in HCV-infected hepatocytes, as suggested by our present results, deserves further investigation.

In summary, taking into account all the findings showed in this paper, it is conceivable that an enhanced LXR-induced hepatic lipogenesis may contribute to lipid accumulation in NAFLD and in chronic hepatitis C infection. However, our results do not support an anti-inflammatory effect of LXR and its dependent transcription factors (PPARγ ) in these chronic liver diseases, likely because their potential antiinflammatory effects are overwhelmed by a number of distinct pro-inflammatory factors, such as lipotoxicity, endoplasmic reticulum stress and mitochondrial dysfunction in NAFLD, and apoptosis and virus-induced production of Th1 cytokines in chronic HCV infection.

In conclusion, this study provides evidence that hepatic expression levels of LXRα and its related lipogenic and inflammatory gene targets are abnormally increased in NAFLD patients and in HCV patients with concomitant fatty liver. These results identify LXRα as a novel factor involved in the pathogenesis of hepatic steatosis in chronic liver diseases. Nevertheless, further experimental studies are needed to establish the specific relationship between LXR and lipogenic and inflammatory pathways in hepatocytes. showing the hepatic protein content of LXRα. Bar graph shows the mean ± SD of the densitometric analysis of specific bands from all patients studied and are expressed as percentage relative to NL (100%). Equal loading of the gels was demonstrated by probing the membranes with an anti-β-actin polyclonal antibody. LXR, liver X receptor; NL, normal liver; NAS, nonalcoholic steatosis; NASH, nonalcoholic steatohepatitis; HCV, hepatitis C virus.*p<0.05 vs NL. # p<0.05 vs NAS. & p<0.05 vs HCV. A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Clinical

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Clinical

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Figure 1 .

 1 Figure 1. Liver biopsy features defining the study population. The degree of steatosis, largely macrovesicular, was similar in patients with steatosis (panel A) and in those with steatohepatitis (panel B). Portal and periportal mononuclear cell infiltrates were common findings in HCV patients without steatosis (panel C) and with steatosis (panel D), showing no significant differences between both patient groups. (All photomicrographs: Hematoxilin-eosin staining; original magnification x200). Magnification bar, 100 µm.

Figure 2 .

 2 Figure 2. Hepatic overexpression of LXRα in NAFLD and HCV patients. (A). Bar graph shows mRNA levels of LXRα determined by real-time RT-PCR in liver biopsies from different patients groups. Values are expressed as mean ± SD of the percentage relative to NL (100%). (B) Representative western blotting (two different patients from each group)showing the hepatic protein content of LXRα. Bar graph shows the mean ± SD of the densitometric analysis of specific bands from all patients studied and are expressed as percentage relative to NL (100%). Equal loading of the gels was demonstrated by probing the membranes with an anti-β-actin polyclonal antibody. LXR, liver X receptor; NL, normal liver; NAS, nonalcoholic steatosis; NASH, nonalcoholic steatohepatitis; HCV, hepatitis C virus.*p<0.05 vs NL. # p<0.05 vs NAS. & p<0.05 vs HCV.

Figure 3 .

 3 Figure 3. Hepatic mRNA levels of LXRα-related lipogenic genes are increased in NAFLD and HCV patients with steatosis. Bar graph shows mRNA levels of PPAR-γ (A), SREBP-1c (B), SREBP-2 (C), and FAS (D) determined by real-time RT-PCR in liver biopsies from different patient groups. Values are expressed as mean ± SD of the percentage relative to NL (100%). PPAR-γ, peroxisome proliferator-activated receptor gamma; SREBP, sterol regulatory element binding protein; FAS, fatty acid synthase; NL, normal liver; NAS, nonalcoholic steatosis; NASH, nonalcoholic steatohepatitis; HCV, hepatitis C virus.*p<0.05 vs NL. # p<0.05 vs NAS. & p<0.05 vs HCV.

Figure 4 .

 4 Figure 4. Hepatic content of PPAR-γ and SREBP-1c proteins is also increased in NAFLD and HCV patients. (A) Representative western blotting (two different patients from each group) showing the hepatic amount of PPAR-γ and SREBP-1c. (B) Bar graph shows the mean ± SD of the densitometric analysis of specific bands from all patients studied and are expressed as percentage relative to NL (100%). Equal loading of the gels was demonstrated by probing the membranes with an anti-β-actin polyclonal antibody. PPAR-γ, peroxisome proliferator-activated receptor gamma; SREBP, sterol regulatory element binding protein; NL, normal liver; NAS, nonalcoholic steatosis; NASH, nonalcoholic steatohepatitis; HCV: hepatitis C virus. *p<0.05 vs NL. # p<0.05 vs NAS. & p<0.05 vs HCV.

Figure 5 .

 5 Figure 5. Hepatic expression of oxidative stress and inflammatory genes is upregulated in NAFLD and HCV patients. Bar graph shows mRNA levels of CYP2E1 (A), OPN (B), TNF-α (C), IL-6 (D) and SOCS-3 (E) determined by real-time RT-PCR in liver biopsies from different patient groups. Values are expressed as mean ± SD of the percentage relative to NL (100%). CYP2E1, cytochrome P450 2E1; OPN, osteopontin; TNF, tumor necrosis factor; IL, interleukin; SOCS, suppressor of cytokine signalling; NL, normal liver; NAS, nonalcoholic steatosis; NASH, nonalcoholic

  steatohepatitis; HCV, hepatitis C virus. *p<0.05 vs NL. # p<0.05 vs NAS. & p<0.05 vs HCV.

Figure 6 .

 6 Figure 6. Hepatic expression of iNOS and COX-2 is markedly induced in NAFLD and HCV patients. Bar graph shows mRNA levels of iNOS (A) and COX-2 (B) determined by real-time RT-PCR in liver biopsies from different patient groups. Values are expressed as mean ± SD of the percentage relative to NL (100%). iNOS, inducible nitric oxide synthase; COX, cyclooxygenase; NL, normal liver; NAS, nonalcoholic steatosis; NASH, nonalcoholic steatohepatitis; HCV, hepatitis C virus.* p<0.05 vs NL. # p<0.05 vs NAS. & p<0.05 vs HCV.

Figure 7 .

 7 Figure 7. Increased hepatic content of OPN, TNF-α and iNOS proteins in NAFLD and HCV patients. (A) Representative western blotting (two different patients from each group) showing the hepatic amount of OPN, TNF-α and iNOS. (B) Bar graph shows the mean ± SD of the densitometric analysis of specific bands from all patients studied and are expressed as percentage relative to NL (100%). Equal loading of the gels was demonstrated by probing the membranes with an anti-β-actin polyclonal antibody. OPN, osteopontin; TNF, tumor necrosis factor; iNOS, inducible nitric oxide synthase; NL, normal liver; NAS, nonalcoholic steatosis; NASH, nonalcoholic steatohepatitis; HCV, hepatitis C virus *p<0.05 vs NL. # p<0.05 vs NAS. & p<0.05 vs HCV.

  Figure 2

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		Clinical Science Immediate Publication. Published on 08 Oct 2010 as manuscript CS20100387
	THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100387	A c c e p t e d M a n u s c r i p t

Table 1 .

 1 ). Equal loading of the gels was demonstrated by probing the membranes with an anti-β-actin polyclonal antibody. OPN, osteopontin; TNF, tumor necrosis factor; iNOS, inducible nitric oxide synthase; NL, normal liver; NAS, nonalcoholic steatosis; NASH, nonalcoholic steatohepatitis; HCV, hepatitis C virus *p<0.05 vs NL. # p<0.05 vs NAS. & p<0.05 vs HCV. Primers and probes used for the multiplex real-time RT-PCR.

			Clinical Science Immediate Publication. Published on 08 Oct 2010 as manuscript CS20100387
		TABLES
		Gene	Genbank	Assay ID	Amplicon size	Dye
		LXRα	NM_005693.2 Hs00172885_m1	78	FAM TM
	THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100387	PPAR-γ SREBP-1c SREBP-2 SOCS-3 FAS CYP2E1 OPN TNF-α IL-6 iNOS COX-2 GAPDH LXRα, liver X receptor alpha; PPAR-γ, peroxisome proliferator-activated receptor NM_015869.4 Hs01115510_m1 92 FAM TM NM_004176.3 Hs00231674_m1 84 FAM TM NM_004599.2 Hs01081778_m1 50 FAM TM NM_003955.3 Hs01000485_g1 111 FAM TM NM_004104 Hs01005611_m1 103 FAM TM NM_000773.3 Hs00559367_m1 71 FAM TM NM_000582.2 Hs00960942_m1 63 FAM TM NM_000594.2 Hs00174128_m1 80 FAM TM NM_000600.2 Hs00985639_m1 66 FAM TM NM_000625.4 Hs00167248_m1 74 FAM TM NM_000963.1 Hs00153133_m1 75 FAM TM NM_002046.3 4326317E 122 VIC TM gamma; SREBP, sterol regulatory element binding protein; SOCS-3, suppressors of cytokine signalling 3; FAS, fatty acid synthase; CYP2E1, cytochrome P450 2E1; OPN, osteopontin; TNF-α, tumor necrosis factor alpha; IL-6, interleukin 6; iNOS, inducible nitric oxide synthase; COX, cyclooxygenase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase A c c e p t e d M a n u s c r i p t

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Table 2 .

 2 Demographic, metabolic, biochemical, viral, and histopathological characteristics of patients with normal liver and nonalcoholic fatty liver disease Clinical Science Immediate Publication. Published on 08 Oct 2010 as manuscript CS20100387

		Characteristics	NL (n=22)	NAS (n=21)	NASH (n=22)
		Age (years)	48.7 ± 11.6	49.1 ± 12.5	48.4 ± 12.1
		Women (%)/Men (%)	14(63.6%) / 8(36.4%)	12(57.1%) / 9(42.9%)	13(59.1%) / 9(40.9%)
		BMI (kg/m 2 )	25.3 ± 2.5	26.0 ± 2.4	26.6 ± 2.1
	THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100387	Glucose (mg/dL) Insulin (µU/L) HOMA-IR TG (mg/dL) LDL-chol (mg/dL) HDL-chol (mg/dL) ALT (IU/L) AST (IU/L) γ-GT (IU/L) Steatosis (%) Grade 0 Grade 1 Grade 2 Grade 3 Fibrosis (%) Stage 0 Stage 1 Stage 2 Stage 3 Stage 4 Necroinflammation (%) Grade 0 Grade 1 Grade 2 Grade 3 Grade 4 Data are shown as mean ± standard deviation or as number of cases (%). NL, 95.2 ± 6.8 96.2 ± 7.4 97.5 ± 6.7 8.5 ± 3.1 10.9 ± 6.6 12.3 ± 4.8* 1.01 ± 0.5 1.45 ± 1.1 1.77 ± 0.9* 107.3 ± 34.3 141.6 ± 46.2* 146.9 ± 45.7* 137.3 ± 38.3 148.1 ± 47.4 141.2 ± 44.1 49.3 ± 8.3 50.7 ± 7.4 48.8 ± 8.7 16.2 ± 6.2 22.1 ± 15.5 49.6 ± 18.2# 19.2 ± 4.6 20.5 ± 9.3 27.3 ± 9.8 28.8± 16.7 61.5 ± 23.6* 73.8 ± 21.5* 22 (100%) 10 (47.7%) 11 (50.0%) 9 (42.8%) 8 (36.4%) 2 (9.5%) 3 (13.6%) 22 (100%) 21 (100%) 4 (18.3%) 10 (45.4%) 7 (31.8%) 1 (4.5%) 22 (100%) 21 (100%) 9 (40.9%) 7 (31.8%) 6 (27.3%) normal liver; NAS, nonalcoholic steatosis; NASH, nonalcoholic steatohepatitis; BMI, body mass index; TG, triglycerides; HOMA-IR, homeostatic model assessment-insulin resistant; LDL-chol, low-density lipoprotein cholesterol; HDL-chol, high-density lipoprotein cholesterol; ALT, alanine aminotransferase; AST, aspartate aminotransferase; γ-GT, gamma-glutamyltransferase. *P < 0.05 with respect to NL group. #P < 0.05 with respect to NAS group. A c c e p t e d M a n u s c r i p t

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Table 3 .

 3 Demographic, metabolic, biochemical, viral, and histopathological characteristics of patients with chronic hepatitis C virus genotype 1 infection Clinical Science Immediate Publication. Published on 08 Oct 2010 as manuscript CS20100387 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100387

	Characteristics	HCV G1 (n=20)	HCV G1+NAS (n=24)
	Age (years)	47.9 ± 11.7		47.2 ± 12.1
	Women (%)/Men (%)	12(60.0%) / 8(40.0%) 14(58.3%) / 10(41.7%)
	BMI (kg/m 2 )	25.8 ± 2.2		26.2 ± 2.6
	Glucose (mg/dL)	95.1 ± 7.7		96.6 ± 8.2
	Insulin (µU/L)	10.1 ± 3.9		10.3 ± 6.9
	HOMA-IR	1.47 ± 0.8		1.52 ± 1.0
	TG (mg/dL)	105.1 ± 44.7		109.8 ± 49.3
	LDL-chol (mg/dL)	139.7 ± 37.4		149.3 ±4 8.2
	HDL-chol (mg/dL)	51.7 ± 8.1		50.6 ± 9.2
	ALT (IU/L)	80.7 ± 26.7		91.6 ± 32.7
	AST (IU/L)	42.2 ± 17.6		51.3 ± 23.5
	γ-GT (IU/L)	85.4 ± 23.0		83.7 ± 24.2
	Viral load (IU/mL)	1.26 ± 0.53 x 10 6		1.43 ± 0.81 x 10 6
	Steatosis (%)
	Grade 0	20 (100%)
	Grade 1				12 (50.0%)
	Grade 2				10 (41.7%)
	Grade 3				2 (8.3%)
	Fibrosis (%)
	Stage 0	5 (25.0%)		4 (16.7%)
	Stage 1	6 (30.0%)		8 (33.3%)
	Stage 2	8 (40.0%)		10 (41.7%)
	Stage 3	1 (5.0%)		2 (8.3%)
	Stage 4
	Necroinflammation (%)
	Grade 0
	Grade 1	8 (40.0%)		11 (45.8%)
	Grade 2	6 (30.0%)		6 (25.0%)
	Grade 3	6 (30.0%)		7 (29.2%)
	Grade 4
	Data are shown as mean ± standard deviation or as number
	of cases (%). HCV G1, hepatitis C virus genotype 1; NAS,
	nonalcoholic steatosis; BMI, body mass index; TG,
	triglycerides; HOMA-IR, homeostatic model assessment-
	insulin resistant; LDL-chol, low-density lipoprotein
	cholesterol;	HDL-chol,	high-density	lipoprotein
	cholesterol; ALT, alanine aminotransferase; AST, aspartate
	aminotransferase; γ-GT, gamma-glutamyltransferase.

© 2010 The Authors Journal compilation © 2010 Portland Press Limited

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A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2010 The Authors Journal compilation © 2010 Portland Press Limited

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& * & * Clinical Science Immediate Publication. Published on 08 Oct 2010 as manuscript CS20100387 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100387 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2010 The Authors Journal compilation © 2010 Portland Press Limited

Acknowledgements

This work was supported by grants to J G-G and S S-C from Junta de Castilla y León (G/467B01/64000/3 and SAN673/LE06/08) and Ministerio de Educación y Ciencia (BFU2007-62977), to JL O from Ayuda a la Investigación Clínica 2009 de Caja Burgos; and to CG-M from Instituto de Salud Carlos III (PI06/0221 and PI10/00067) and Fundación Eugenio Rodríguez Pascual. MV G-M and TL-R were supported by CIBERehd contracts. CIBERehd is funded by the Instituto de Salud Carlos III, Spain.

Author contribution

All the listed authors have participated actively in the study.