Recombinant human manganese superoxide dismutase reduces liver fibrosis and portal pressure in CCl4-cirrhotic rats

Research Article

Recombinant human manganese superoxide dismutase reduces liver fibrosis and portal pressure in CCl4-cirrhotic rats Maeva Guillaume1, , Aina Rodriguez-Vilarrupla1, , Jorge Gracia-Sancho1, Eugenio Rosado1, Aldo Mancini2, Jaume Bosch1, Joan Carles Garcia-Pagán1,⇑ 1

Hepatic Haemodynamic Laboratory, Liver Unit, Hospital Clínic, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS) and CIBERehd, University of Barcelona, Spain; 2Department of Molecular Biology and Viral Oncogenesis, Istituto Nazionale dei Tumori of Naples, Naples, Italy

Background & Aims: High oxidative stress plays a major role in increasing hepatic vascular resistance in cirrhosis, by facilitating liver fibrosis and by increasing hepatic vascular tone. This study is aimed at investigating whether the use of the novel isoform of recombinant human manganese superoxide dismutase (rMnSOD) could be a new therapeutic strategy to reduce oxidative stress and portal hypertension in cirrhotic rats. Methods: In CCl4- and BDL-cirrhotic rats treated with rMnSOD (i.p. 15 lg/kg/day) or its vehicle for 7 days, mean arterial pressure (MAP), portal pressure (PP) and portal blood flow (PBF) or small mesenteric arterial flow (SMABF) were measured. In addition, in CCl4-cirrhotic rats, we evaluated the hepatic vasodilatory response to acetylcholine, liver fibrosis with Sirius red staining and hepatic stellate cell activation by a-smooth muscle actin (a-SMA) protein expression. Results: rMnSOD treatment significantly reduced PP either in CCl4- or BDL-cirrhotic rats without significant changes in splanchnic blood flow, suggesting a reduction in hepatic vascular resistance. MAP was not modified. Reduction in PP was associated with a significant reduction in liver fibrosis, and a-SMA protein expression as well as with improved vasodilatory response to acetylcholine. Conclusions: Chronic rMnSOD administration to cirrhotic rats reduces portal pressure by reducing hepatic vascular resistance without deleterious effects on systemic hemodynamics, suggesting that it might constitute a new antioxidant to be considered as additional therapy for treating portal hypertension in cirrhosis.

Received 26 March 2012; received in revised form 5 September 2012; accepted 7 September 2012; available online 16 September 2012 ⇑ Corresponding author. Address: Hepatic Haemodynamic Laboratory, Liver Unit, Hospital Clínic, Villarroel 170, 08036 Barcelona, Spain. Tel.: +34 93 227 5400x2824; fax: +34 93 227 9856. E-mail address: [email protected] (J.C. Garcia-Pagán).   These authors contributed equally to this work and share first authorship. Abbreviations: a-SMA, a-smooth muscle actin; Ach, acetylcholine; AU, arbitrary units; CCl4, carbon tetrachloride; DHE, dihydroethidium; HSC, hepatic stellate cells; HRP, horseradish peroxidase; IVR, intrahepatic vascular resistance; LSEC, liver sinusoidal endothelial cells; MAP, mean arterial pressure; Mtx, methoxamine; NO, nitric oxide; O 2 , superoxide; PBF, portal blood flow; PP, portal pressure; rMnSOD, recombinant human manganese-containing superoxide dismutase; SOD, superoxide dismutase.

Ó 2012 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Introduction Increased intrahepatic vascular resistance is the primary factor in the development of portal hypertension, the main complication of cirrhosis, and it is the result of both structural changes in the liver that are inherent to cirrhosis and increased hepatic vascular tone [1–3]. Sinusoidal endothelial dysfunction, characterized by impaired endothelium-dependent vasodilatation, is a major pathogenic factor in the increased vascular tone and is mainly attributed to insufficient nitric oxide (NO) availability within the cirrhotic liver. The decreased liver NO is due to diminished NO synthase (eNOS) activity [4–6] and to NO scavenging by increased release of superoxide (O 2 ) [7]. We have previously shown that the increased O 2 content in cirrhotic livers is a consequence not only of increased production but also of decreased expression and activity of superoxide dismutase (SOD), the critical antioxidant enzyme in the liver [8]. Our group has clearly demonstrated that strategies which decrease intrahepatic O 2 levels enhance intrahepatic NO bioavailability and reduce portal pressure in portal hypertensive cirrhotic rats [9]. Two different experimental therapeutic strategies have previously been evaluated, adenoviral delivery of SOD [10] and intraportal administration of tempol (a SOD mimetic) [11]. However, the use of adenovirus-based gene therapies is highly controversial in humans, and although tempol administration is known to reduce portal pressure it may also have systemic effects. Recently, a novel recombinant isoform of human manganese SOD (rMnSOD) has been developed. The particular property of this form of SOD is that after administration, it enters cells, due to its uncleaved terminal peptide sequence [12–14]. It has been shown to be very effective at scavenging intra and extracellular O 2 and at improving pathological conditions associated with increased oxidative stress [15,16]. In addition, rMnSOD shows good biodistribution in the liver compared to other organs, suggesting that it is well suited for correcting hepatic oxidative stress [13]. The present study therefore aimed at investigating whether chronic administration of rMnSOD could be a new therapeutic strategy to reduce portal hypertension in cirrhosis.

Journal of Hepatology 2013 vol. 58 j 240–246

JOURNAL OF HEPATOLOGY Materials and methods Effects of rMnSOD on the increased hepatic O 2 levels promoted by NADPH oxidase To confirm that rMnSOD (produced in the lab of Dr. Aldo Mancini, Istituto Nazionale dei Tumori di Napoli, Naples, Italy) [12–15] is able to scavenge increased intrahepatic O 2 levels, control rats received a single dose of rMnSOD (15 lg/kg, i.p.) or vehicle (phosphate buffer saline, PBS) 2 h before the experiment. Afterwards, livers were isolated and perfused as described below, and oxidative stress was generated by adding nicotinamide adenine dinucleotide phosphate (NADPH; 1 mM; Applichem, Darmstadt, Germany), the substrate of NADPH oxidase, to the perfusion system, as previously described [10]. After 10 min, rat livers were promptly removed and in situ O 2 content was evaluated in fresh liver cryosections (10 lm, n = 4 per group) using the dye dihydroethidium (DHE; 10 lM) (Molecular Probes Inc., Eugene, OR), as previously described [7]. Six fluorescence images per cryosection were obtained with a laser scanning confocal microscope (TCS-SL DMIRE2, Leica) and quantitative analyses were performed using Image J 1.43u software (National Institutes of Health (NIH), USA). Induction of cirrhosis by carbon tetrachloride (CCl4) Male Wistar rats (weighing 50–75 g) underwent repeated inhalation exposure to CCl4. Phenobarbital (0.3 g/L) was added to their drinking water as previously described [17,18]. After approximately 12–16 weeks, the animals developed ascites and CCl4 and phenobarbital administration was discontinued. One week later, animals were distributed randomly into two groups (n = 9 for Vehicle and n = 11 for rMnSOD). Induction of cirrhosis by common bile duct ligation (BDL) Secondary biliary cirrhosis with intrahepatic portal hypertension was induced in male Sprague–Dawley rats (250–275 g) by common bile duct ligation (BDL) as described [19]. While each animal was under anesthesia, the common bile duct was occluded by double ligature with 5-0 silk thread. The bile duct was then resected between the two ligatures. Two weeks later, animals were randomly divided into two groups (n = 12 for Vehicle and n = 14 for rMnSOD). rMnSOD treatment rMnSOD (15 lg/kg body weight, i.p.) or its vehicle (PBS) was administered daily for 7 days to CCl4- and BDL-cirrhotic rats. rMnSOD or its vehicle was prepared by a third person, and therefore the investigators administering the drug and performing the experiments were not aware of the treatment received by the rats, which was kept under code until the final analysis of results. Final experiments were performed 2 h after the last injection. The dose of rMnSOD administered has been shown to protect against oxidative stress in other pathologies [13– 16]. Animals were kept in environmentally-controlled animal facilities at the IDIBAPS (Institut d’Investigacions Biomèdiques August Pi i Sunyer). All experiments were approved by the Laboratory Animal Care and Use Committee of the University of Barcelona and were conducted in accordance with European Community guidelines for the protection of animals used for experimental and other scientific purposes (EEC Directive 86/609). Immunohistochemistry for rMnSOD

the competition reaction between the sample-containing SOD and the highly water-soluble tetrazolium salt (WST) that produces a water-soluble formazan dye upon reaction with O 2 . Briefly, livers were homogenized in buffer containing 20 mM Hepes, 1 mM EDTA, 210 mM mannitol and 70 mM sucrose. After centrifugation at 1500g for 5 min at 4 °C, the supernatant was collected and the protein concentration quantified. The SOD activity assay was performed according to manufacturer instructions. Hepatic O 2 levels were quantified using the SOD activity assay with minor modifications. Briefly, 20 lg of protein for each liver sample (n = 7 per group) was incubated with WST for 20 min at 37 °C. Positive (exogenous O 2 generating enzyme) and negative (samples with high antioxidant capacity) internal controls were included. In vivo haemodynamic studies Cirrhotic rats were anaesthetized with an intraperitoneal injection of ketamine (100 mg/kg body weight, Merial, Barcelona, Spain) plus midazolam (5 mg/kg body weight, Laboratorio Reig Jofré, Barcelona, Spain) and maintained at a constant temperature of 37 ± 0.5 °C during surgery. After tracheotomy with a PE240 catheter (Portex, Kent, UK), PE-50 catheters (Portex) were introduced into the femoral artery and the ileocolic vein to measure mean arterial pressure (MAP, mmHg) and portal pressure (PP, mmHg), respectively [20]. Perivascular ultrasonic flow probes connected to a flow meter (Transonic Systems Inc., Ithaca., NY, USA) were placed in the CCl4-cirrhotic rats, around the portal vein as close as possible to their origins (to avoid portal-collateral blood flow) in order to measure portal blood flow (PBF, mlmin1), and, in BDL-cirrhotic rats, at the superior mesenteric artery to measure superior mesenteric artery blood flow (SMABF; mlmin1). Haemodynamic data were collected after 20 min of stabilization and registered on a multichannel computer-based recorder (PowerLab, 8SP) using the Chart v5.0.1 for Windows software (AD Instruments, Mountain View, LA). Intrahepatic vascular resistance (IVR) was estimated as PP/PBF (mmHg/ ml min1). Biochemical analysis At the end of the in vivo haemodynamic study, serum samples from CCl4-cirrhotic rats were collected from the femoral artery to subsequently evaluate alanine aminotransferase (ALT), aspartate aminotransferase (AST), and albumin, all by current standard protocols. Evaluation of endothelial function After in vivo haemodynamic measurements in CCl4-cirrhotic rats, livers were quickly isolated and perfused by a flow-controlled perfusion system, as previously described [6,18]. Following 20 min of stabilization, intrahepatic microcirculation was preconstricted by adding the a1-adrenergic agonist methoxamine (Mtx; 104 mol/L; Sigma) to the reservoir. After 5 min, endothelial function was evaluated by the vasodilatory response to cumulative doses of acetylcholine (Ach, 107, 106, 105 mol/L; Sigma) administered every 1.5 min. Responses to acetylcholine were calculated as the percentage change in portal perfusion pressure (PPP). Viability and stability criteria of liver perfusion preparation included gross appearance of the liver, stable perfusion pressure, bile production over 0.4 ll/min/g liver, and a stable buffer pH (7.4 ± 0.3) during the initial 20 min stabilization period [18]. If either viability or stability criteria were not satisfied, the experiment was discarded.

To confirm that rMnSOD actually got into treated rat livers, immunostaining of 5 paraffin-embedded liver sections from CCl4-cirrhotic rats was performed with a rabbit polyclonal antibody produced by Dr. Mancini and directed against its unique uncleaved terminal sequence specific for rMnSOD [12–14]. Briefly, sections were treated twice with PBS containing 0.3% hydrogen peroxide and incubated with the primary antibody (1/100, 30 min, RT). Bound antibodies were visualized using diaminobenzidine as the chromogen, and slides were then counterstained with hematoxylin solution for 10 min before being mounted and examined using light microscopy (Zeiss Axiovert) for a qualitative analysis [7]. For the negative control, PBS was used instead of the primary antibody.

Livers from CCl4-cirrhotic rats treated with rMnSOD (n = 8) or vehicle (n = 8) were fixed in 10% formaldehyde, embedded in paraffin, sectioned, and stained with 0.1% Sirius red for semiquantitative analysis of hepatic fibrosis [19,21,22]. Eight images of each sample were obtained at a magnification of 5 with an inverted optical microscope (Zeiss Axiovert, Germany) equipped with a digital camera and then measured using AxioVision software. The red-stained area per total area was quantified using a morphometric method.

Effects of rMnSOD on hepatic SOD activity and oxidative stress

Protein expression of a-smooth muscle actin (aSMA)

Total SOD activity was measured in liver homogenates obtained from CCl4cirrhotic rats treated with rMnSOD or vehicle (n = 9 per group) using a commercially available immunoassay (Sigma, Tres Cantos, Madrid). The assay is based on

Hepatic a-SMA protein expression was determined by Western blot, as previously described [23], using a mouse anti-a-SMA primary antibody (1/ 1000, overnight, 4 °C; Sigma), followed by incubation with a rabbit anti-mouse

Effects of rMnSOD on hepatic fibrosis

Journal of Hepatology 2013 vol. 58 j 240–246

241

Research Article A

Superoxide levels

A.U. normalized to vehicle

HRP-conjugated secondary antibody (1:10,000; Stressgen, Victoria, BC, Canada). Blots were revealed by chemiluminescence. Quantitative densitometric values of a-SMA determined using the Science Lab 2001 Image Gauge software (Fuji Photo Film Gmbh, Düsseldorf) were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Effects of rMnSOD on ROS and SMA and procollagen I gene expression in hepatic stellate cells

Vehicle

rMnSOD

B

Vehicle

rMnSOD

rMnSOD (negative control)

Statistics

rMnSOD abolishes O 2 levels promoted by NADPH oxidase in control rat livers Control rats pre-treated with a single i.p. dose of rMnSOD, 2 h before activation of the NADPH/NADPH oxidase system, exhibited significantly reduced levels of intrahepatic O 2 in comparison to rats pre-treated with vehicle (87% reduction; p <0.016; Fig. 1A), demonstrating that rMnSOD reaches the liver where it is functionally active. Determination of rMnSOD presence and activity in cirrhotic rat livers Livers from CCl4-cirrhotic animals receiving human rMnSOD showed positive staining for the protein, whereas this was not observed in vehicle-treated rats (Fig. 1B top panel). To evaluate whether intrahepatic rMnSOD is able to scavenge intrahepatic

p = 0.05

1.2 1.1 1.0 0.9 0.8 0.7 0.6 0.5

p = 0.06

Ve h rM icle nS O D

n=9

Superoxide levels

A.U. normalized to vehicle

There were no deaths during treatment in neither CCl4- nor BDLcirrhotic rats. There were no significant differences between CCl4cirrhotic rats treated with vehicle or rMnSOD in terms of AST, ALT or albumin (Table 1).

1.3 1.2 1.1 1.0 0.9 0.8 0.7 0.6 0.5

Ve h rM icle nS O D

Results

Total SOD activity

A.U. normalized to vehicle

All analyses were performed with SPSS 19.0 for Windows (SPSS Inc., Chicago, IL). Results were expressed as mean ± SD. Comparisons between groups were performed using the Student’s t test for unpaired data, or Mann–Whitney test when assumptions of normality could not be verified (Kolmogorov–Smirnov test). Significance was set at the 0.05 level.

p = 0.016

Ve h rM icle nS O D

To directly study the effect of rMnSOD treatment on HSC, we used the immortalized humane stellate cell line (LX-2 cells; kindly provided by Dr. Bataller), which was previously well characterized [24]. LX-2 were seeded onto p35 plates at density of two hundred fifty thousand per plate in Dulbecco’s minimal essential medium supplemented with 10% fetal bovine serum, 1% penicillin, and 1% streptomycin and cultured overnight. rMnSOD was added at concentration of 1 lM to subconfluent cultures of LX-2 for 24 h. Superoxide levels were assessed with the oxidative fluorescent dye DHE as previously described [7]. a-SMA and procollagen I gene expression were determined by real time PCR using predesigned gene expression assays obtained from Applied Biosystems according to the manufacturer’s protocol and reported relative to endogenous control 18S. All reactions were performed in duplicate and using nuclease-free water as no template control.

1.2 1.0 0.8 0.6 0.4 0.2 0.0

n=7

Fig. 1. Localization and function of rMnSOD on increased hepatic O 2 levels. (A) Effects of rMnSOD on the increased hepatic O 2 levels promoted by NADPH oxidase. (Left panel) Representative confocal fluorescence microscopy images of in situ detection of O 2 with dihydroethidium (DHE) in fresh liver sections from control rats treated with an i.p. injection of rMnSOD or vehicle in response to nicotinamide adenine dinucleotide phosphate (NADPH). (Right panel) DHE fluorescence intensity analysis showed a marked and significant reduction in intrahepatic O 2 in control rats treated with rMnSOD (87% reduction vs. vehicletreated rats; p <0.016). Data are presented as arbitrary units (A.U.) normalized to vehicle ±SEM. (B) Determination of rMnSOD presence and activity in cirrhotic rat livers. (Top panel) Representative histological images of livers from cirrhotic rats treated with vehicle or rMnSOD after immunohistochemical reaction with a specific antibody against the unique uncleaved terminal sequence specific for rMnSOD (original magnification 10). Presence of rMnSOD was detected by a brown staining in the liver interstitial space and in the cytoplasm of hepatocytes and endothelial cells. Absence of reaction appeared in blue, as shown in the negative control (in this case, PBS was used instead of the primary antibody). (Bottom left panel) Total SOD determined in liver homogenates from cirrhotic rats treated with rMnSOD or vehicle (18% increase; p = 0.05). (Bottom right panel) Superoxide levels were significantly decreased in rMnSOD-treated rat livers (26% decrease; p = 0.06). Data are expressed as arbitrary units (A.U.) normalized to vehicle ±SEM. SOD, total superoxide dismutase.

Table 1. Effects of rMnSOD treatment on liver function.

Vehicle (n = 9)

rMnSOD (n = 11)

p value

AST (U/L)

211 ± 73

169 ± 68

0.20

ALT (U/L)

73 ± 22

71 ± 19

0.87

Albumin (g/L)

25.2 ± 8

28.3 ± 5

0.32

Data are presented as mean ± SD. AST, aspartate aminotransferase; ALT, alanine aminotransferase.

242

O 2 , we determined total SOD activity in hepatic homogenates. As expected, total SOD activity was increased (+18%; p = 0.05; Fig. 1B bottom left panel) in livers from cirrhotic rats treated with rMnSOD compared to those treated with vehicle. This was associated with a decrease in O 2 levels in livers from cirrhotic rats treated with rMnSOD compared to vehicle (26% decrease, p = 0.06; Fig. 1B bottom right panel).

Journal of Hepatology 2013 vol. 58 j 240–246

JOURNAL OF HEPATOLOGY rMnSOD (n = 11) 12.6 ± 1.9 3.6 ± 1.1 1.0 ± 0.4 82 ± 21 351 ± 42 361 ± 43

p value 0.02 0.21 0.03 0.58 0.37 0.82

rMnSOD treatment significantly reduced PP (mean decrease 14%) and IVR in CCl4-cirrhotic rats. Body weight, PBF and systemic (MAP and HR) haemodynamics did not differ between the groups. Data are presented as mean ± SD. PP, portal pressure; PBF, portal blood flow; IVR, intrahepatic vascular resistance; MAP, mean arterial pressure; HR, heart rate.

20 10 0

-20

p = 0.002 p = 0.004 p = 0.005

-30 Pre-Ach

-7

PP (mmHg) SMABF (ml/min/100g bw) SAR (mmHg/ml/min) MAP (mmHg) HR (beat/min) Body weight (g)

rMnSOD (n = 14) 13.1 ± 1.9 2.5 ± 0.7 7.7 ± 3.5 84 ± 17 325 ± 19 394 ± 40

B

1.00

eNOS

Vehicle rMnSOD

rMnSOD treatment significantly reduced PP (mean decrease 14%) in BDL-cirrhotic rats. Body weight, and systemic (MAP, SMABF, SAR and HR) haemodynamics did not differ between the groups. Data are presented as mean ± SD. PP, portal pressure; SMABF, small mesenteric artery blood flow; SAR, small arteriolar resistance; MAP, mean arterial pressure; HR, heart rate.

rMnSOD reduces portal pressure and intrahepatic vascular resistance in cirrhotic rats CCl4-cirrhotic rats treated with rMnSOD exhibited significantly lower portal pressure than did those receiving vehicle (12.6 ± 1.9 mmHg in rMnSOD vs. 14.7 ± 1.7 mmHg in Veh; mean decrease 14%; p = 0.022), there being no significant differences in MAP or PBF. The reduction in PP was associated with a significant decrease in intrahepatic vascular resistance (Table 2). Similarly, portal pressure was significantly lower in BDLcirrhotic rats treated with rMnSOD in comparison to those treated with vehicle (13.1 ± 1.9 mmHg in rMnSOD vs. 15.3 ± 2.1 mmHg in Veh; mean decrease 14%; p = 0.015) without significant changes in MAP or SMABF (Table 3). rMnSOD improves hepatic endothelial dysfunction in cirrhotic rats Baseline portal perfusion pressure was significantly lower in CCl4-cirrhotic rats treated with rMnSOD than in those treated with vehicle (7.9 ± 2.1 mmHg in rMnSOD vs. 10.1 ± 1.6 mmHg in Veh; mean decrease 21%; p = 0.032). As expected, cirrhotic livers treated with vehicle exhibited marked endothelial dysfunction. rMnSOD treatment significantly improved vasorelaxation in response to Ach (Fig. 2A). To explore whether the improvement of endothelial dysfunction was associated with an amelioration of NO pathway, we

0.75 0.50 0.25 0.00

p value 0.01 0.26 0.83 0.22 0.62 0.84

-5

Ve h rM icle nS O D

Vehicle (n = 12) 15.2 ± 2.1 2.8 ± 0.9 7.4 ± 2.8 92 ± 15 333 ± 56 397 ± 34

-6

Ach (log10 M)

GAPDH Table 3. Effects of rMnSOD treatment on systemic, splanchnic and hepatic haemodynamics in BDL-cirrhotic rats.

Vehicle rMnSOD

-10

cGMP levels (pmol/mg tissue)

PP (mmHg) PBF (ml/min/100g bw) IVR (mmHg/ml/min) MAP (mmHg) HR (beat/min) Body weight (g)

Vehicle (n = 9) 14.7 ± 1.7 2.9 ± 1.0 1.7 ± 0.7 87 ± 17 368 ± 43 356 ± 57

A

Change in PP (%)

Table 2. Effects of rMnSOD treatment on systemic, splanchnic and hepatic haemodynamics in CCl4-cirrhotic rats.

Fig. 2. Effect of rMnSOD treatment on endothelial function in CCl4-cirrhotic rats. (A) Endothelium-dependent vasorelaxation to acetylcholine (Ach) in isolated and perfused livers of CCl4-cirrhotic rats treated with rMnSOD (n = 10) or vehicle (n = 8). Results are expressed as the percentage change in PP in response to Ach and are presented as mean ± SEM. rMnSOD treatment significantly improved the impaired vasodilatory response to Ach in CCl4-cirrhotic rat livers. PP, portal perfusion pressure. (B) (Left panel) eNOS expression. Representative Western blot analysis for eNOS in livers from Veh- or rMnSOD-treated CH rats. Densitometric quantifications in arbitrary units (A.U.) normalized to GADPH are shown. All results are expressed as mean ± SEM normalized to vehicle. (Right panel) Intrahepatic cGMP levels in CH rats treated with rMnSOD or vehicle. Values are expressed as pmol/mg tissue. No significant differences in cGMP levels were observed (n = 8 per group).

determined cGMP levels and eNOS protein expression in livers from CCl4-cirrhotic rats treated with vehicle or rMnSOD. No differences in cGMP content (a marker of NO bioavailability) or in eNOS expression were observed among both groups (Fig. 2B). rMnSOD attenuates hepatic fibrosis in CCl4-cirrhotic rats and deactivates hepatic stellate cells in vitro Livers from CCl4-cirrhotic animals receiving vehicle exhibited marked architectural distortion, with extensive deposition of fibrillar collagen and small regenerating nodules (Fig. 3A, top panel). Livers from rats receiving rMnSOD showed a 51.8% reduction in collagen deposition (p <0.0001; Fig. 3A, bottom panel), resulting in thinner septa and larger regenerating nodules compared to those from rats treated with vehicle (Fig. 3A, top panel). This was associated with a marked decrease in a-SMA expression, a marker of activated hepatic stellate cells (76% less a-SMA expression than in livers from vehicle-treated rats; p = 0.012; Fig. 3B). To further corroborate the role of rMnSOD on hepatic stellate cells, we performed in vitro experiments with LX2 cells. rMnSOD promoted an evident decline in basal O 2 content in cultured LX2. This reduction in oxidative stress was associated with a pronounced reduction of a-SMA and collagen I gene expression (Fig. 4). Taken together, these results confirmed that rMnSOD treatment markedly reduced hepatic stellate cell activation and hepatic fibrosis.

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Research Article A

A

rMnSOD

B

11

rMnSOD

1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0

1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0

p <0.05

n=6

Fig. 3. Effects of rMnSOD treatment on intrahepatic fibrosis in CCl4-cirrhotic rats. (A) (Top panel) Histological images of livers stained with Sirius red from cirrhotic rats treated with vehicle or rMnSOD. Representative sections are shown at original magnification 5. (Bottom panel) Quantification of liver fibrosis (Sirius red staining area per total area) from a mean of 8 pictures for each slide. (B) aSMA expression. (Left panel) Representative Western blot analysis for a-SMA in livers from Veh- or rMnSOD- treated CH rats. (Right panel) Densitometric quantifications in arbitrary units (A.U.) normalized to GADPH are shown. rMnSOD treatment significantly reduced intrahepatic fibrillar collagen (51.8%, p <0.0001) and a-SMA expression (76%, p = 0.012), a surrogate marker of hepatic stellate cells activation. All results are expressed as mean ± SEM normalized to vehicle.

Discussion Increased intrahepatic resistance within the cirrhotic liver, the primary cause of portal hypertension, is mainly due to increased intrahepatic resistance, which results from structural changes inherent to progressive fibrosis and dynamic changes due to increased hepatic vascular tone [1–3]. Low NO bioavailability and exacerbated production of vasoconstrictors, that leads to the contraction of hepatic stellate cells, are considered the main factors responsible for endothelial dysfunction, and together with hepatic fibrosis, they represent the most obvious therapeutic targets for improving portal hypertension [9]. Indeed, as elevated oxidative stress in the cirrhotic liver is one of the mechanisms leading to both liver fibrosis and reduced NO availability [7], several attempts have been made to reduce hepatic oxidative stress in cirrhotic animal models. 244

p <0.05

Fig. 4. Effects of rMnSOD treatment on hepatic stellate cells in vitro. (A) Representative confocal fluorescence microscopy images of in situ detection of superoxide with the oxidative dye dihydroethidium (DHE) in LX-2 treated with vehicle or rMnSOD for 24 h. (B) Relative a-SMA and collagen I mRNA levels in LX-2 treated with vehicle (n = 12) or rMnSOD (n = 12) normalized to an endogenous reference gene (18S). Values (mean ± SEM) are normalized to Veh-treated LX2 cells.

p = 0.012

Ve h rM icl nS e O D

Vehicle

Collagen I expression

α-SMA expression

A.U. normalized to GAPDH

α-SMA GAPDH

1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0

Ve h rM icle nS O D

=

B

α-SMA expression

A.U. normalized to vehicle

p <0.05

Ve h rM icle nS , n O D =8 ,n

A.U. normalized to vehicle

1.2 1.0 0.8 0.6 0.4 0.2 0.0

rMnSOD

Ve h rM icle nS O D

Vehicle Quantification of liver fibrosis (sirius red/total area)

A.U. normalized to vehicle

Vehicle

Detection of superoxide

Recently, a novel isoform of human MnSOD has been identified. Here we hypothesized that the chronic administration of rMnSOD to cirrhotic animals might decrease hepatic oxidative stress, improve intrahepatic vascular resistance, and ultimately, decrease portal pressure. To validate our hypothesis, we firstly characterized rMnSOD distribution and activity within the liver after injection of the protein. Results from these experiments demonstrated that the acute i.p. administration of rMnSOD to control rats results in a marked reduction of the hepatic oxidative stress generated experimentally through activation of the NADPH-oxidase system, confirming its biodistribution in the liver and the preservation of enzymatic activity. More importantly, after the chronic administration of rMnSOD to cirrhotic animals, we demonstrated its presence in the liver, which led to increased hepatic SOD activity and efficiently decreased the elevated levels of O 2 . This indicates that rMnSOD may represent a novel therapeutic option for liver diseases in which increased oxidative stress plays a pathogenic role [12–14]. Once the therapeutic agent had been validated in our experimental model, the possible effects of rMnSOD on hepatic and systemic haemodynamics were analyzed in two models of cirrhosis: CCl4 and BDL. Our study clearly demonstrates that rMnSOD significantly reduced portal pressure in vivo without modifying systemic or splanchnic haemodynamics in both models of cirrhosis. The magnitude of PP reduction averaged 14%, which is the most pronounced reduction in PP observed using antioxidant strategies in cirrhotic rats [10,11,25,26]. The decrease in PP was not associated with changes in portal blood flow, therefore suggesting a significant reduction in hepatic vascular resistance.

Journal of Hepatology 2013 vol. 58 j 240–246

JOURNAL OF HEPATOLOGY To identify the mechanisms of rMnSOD-derived improvement in intrahepatic resistance, we characterized hepatic fibrosis status and hepatic endothelial function in CCl4-cirrhotic rats. Hepatic fibrosis was studied by analyzing fibrillar collagen content and the expression of a-SMA, a marker of hepatic stellate cell activation. We observed that rMnSOD-treated rats exhibited a marked decrease in liver fibrillar collagen (mean decrease 51.8%), with thinner septa and larger regenerating nodules compared to those of rats treated with vehicle. It is important to emphasize that greater septal thickness and smaller nodule size have been associated with a more severe degree of portal hypertension [27]. In addition, rMnSOD treatment significantly reduced a-SMA expression (mean decrease 76%). These results, indicating a clear antifibrotic effect, are in accordance with a previous report showing hepatocyte protection and fibrosis attenuation due to antioxidant-derived hepatic stellate cell de-activation [28]. In accordance with that, our results on LX-2 cells show that rMnSOD is able to scavenge basal O 2 content and significantly decrease SMA and collagen I gene expression suggesting HSC deactivation. In association with this improvement in the architectural alterations of the liver, we found that rMnSOD-treated cirrhotic rats have a significantly improved hepatic endothelial function. In fact, the well-known paradoxical vasoconstriction of cirrhotic livers in response to increasing doses of acetylcholine was no longer observed in animals treated with rMnSOD. These beneficial effects of rMnSOD on the hepatic endothelial function could be related to improved NO availability associated with O 2 reduction, as previously demonstrated by our group and others [7,10,11,25,26]. In conflict with this mechanism, we are unable to detect a significant increase in hepatic cGMP levels, a surrogate of NO bioavailability. However, we cannot rule out that the cGMP technique was not sensitive enough to sense small increases in NO bioavailability as those expected by the observed subtle reduction in O 2 levels, far lower than that observed with other antioxidant strategies that we have previously used [10,11]. It is also conceivable that the improvement in liver fibrosis plays a major role in the recovery of the endothelial function. It is worth mentioning that rMnSOD did not decrease the systemic arterial pressure, which represents a clear advantage of the current strategy in the treatment of cirrhotic portal hypertension. In conclusion, the current study demonstrates that the chronic administration of rMnSOD to cirrhotic rats markedly decreases hepatic vascular resistance, mainly by ameliorating liver fibrosis leading to a significant reduction in portal pressure. Our study suggests that rMnSOD may represent a useful agent in the treatment of patients with cirrhosis of the liver.

Financial support Maeva Guillaume was supported by the Direction pour la Recherche Clinique et l’Innovation de Toulouse. This study was supported by grants from the Ministerio de Educación y Ciencia (SAF 2010/17043) and from the Instituto de Salud Carlos III (PS09/0126, ACI2009-0938 and FIS PI11/00235), Spain. J.G.-S. has a contract from the Programa Ramón y Cajal, Ministerio de Economía y Competitividad, Spain. CIBERehd is funded by the Instituto de Salud Carlos III. Part of this work was carried out at the Esther Koplowitz Centre, Barcelona.

Conflict of interest The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. Acknowledgements Part of this work was carried out at the Esther Koplowitz Centre, Barcelona. The authors would like to thank Hector Garcia, Montse Monclús, Antonella Borrelli, Antonella Schiattarella and Roberto Mancini for their technical assistance. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jhep.2012. 09.010.

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