Liver sinusoidal endothelial dysfunction after LPS administration: A role for inducible-nitric oxide synthase

Research Article

Liver sinusoidal endothelial dysfunction after LPS administration: A role for inducible-nitric oxide synthase Vincenzo La Mura1,2, , Marcos Pasarín1, , Aina Rodriguez-Vilarrupla1, Juan Carlos García-Pagán1, Jaime Bosch1, Juan G. Abraldes1,3,⇑ 1

Hepatic Hemodynamic Laboratory, Liver Unit, Hospital Clínic-IDIBAPS, University of Barcelona, and Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (Ciberehd), Spain; 2Fondazione IRCCS Ca’ Granda, Ospedale Maggiore Policlinico, Università di Milano, Milano, Italy; 3Division of Gastroenterology, Department of Medicine, University of Alberta, Edmonton, Canada

Background & Aims: Sepsis is associated with microvascular dysfunction, which contributes to organ failure. Intrahepatic endothelial dysfunction occurs after exposure to lipopolysaccharide (LPS). The upregulation of inducible nitric oxide synthase (iNOS) has been shown to contribute to systemic vascular dysfunction after LPS administration. However, little is known about the effects of iNOS induction on the liver microcirculation. This study aimed at exploring, in the isolated rat liver perfusion model, the role of iNOS induction in liver microvascular dysfunction associated with endotoxemia. Methods: All experiments were conducted in male Wistar rats, after 24 h of LPS (5 mg/kg i.p.) or saline administration in the presence or absence of the iNOS inhibitor 1400W (3 mg/kg i.p.), administered 3 and 23 h after LPS/saline injection. Liver microvascular function was assessed by isolated liver perfusion, followed by molecular studies and liver function tests. Results: At 24 h, LPS induced liver endothelial dysfunction, as shown by a decreased vasodilatory response to acetylcholine and decreased eNOS phosphorylation at Ser1176. This was associated with liver injury, assessed by an increase in liver transaminases and decreased indocyanin green clearance, and increased nitrooxidative stress. iNOS inhibition prevented liver endothelial dysfunction, blunted the development of liver injury and attenuated LPS-induced nitrooxidative stress. Conclusions: iNOS upregulation contributes to liver microvascular dysfunction in endotoxemia. This suggests that this mechanism deserves further exploration in studies addressing liver protection in the context of severe acute bacterial infection. Ó 2014 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. Keywords: Sepsis; 1400W; Oxidative stress; Sinusoidal endothelial cells; Multi organ failure. Received 4 February 2014; received in revised form 4 July 2014; accepted 4 July 2014; available online 16 July 2014 ⇑ Corresponding author. Address: Division of Gastroenterology (Liver Unit), University of Alberta, 1-51 Zeidler Ledcor Building, Edmonton, Alberta T6G-2X8, Canada. Tel.: +34 656568321. E-mail address: [email protected] (J.G. Abraldes).   These authors contributed equally to this work. Abbreviations: LPS, lipopolysaccharide; MOF, multi-organ failure; iNOS, inducible nitric oxide synthase; eNOS, endothelial NOS; ICG, indocyanine green; ED, endothelial dysfunction; DHE, dihydroethidium; PPP, portal perfusion pressure; BH4, tetrahydrobiopterin.

Introduction Liver dysfunction is one of the earliest high-risk markers for multi-organ failure (MOF) in patients with sepsis [1]. The integrity of the liver microvasculature, with an adequate function of sinusoidal endothelial cells, plays a fundamental role in maintaining liver perfusion and liver cell viability [2]. In a recent study, using a murine model, we demonstrated that lipopolysaccharide (LPS) injection provokes hepatic microvascular dysfunction, associated with marked endothelitis in perisinusoidal areas [3]. Furthermore, we demonstrated that simvastatin, a drug that improves liver endothelial dysfunction [4–6], ameliorated liver injury and liver inflammation induced by LPS [3]. It is well established that endotoxemia is associated with the upregulation of inducible endothelial nitric oxide synthase (iNOS) [7]. Studies on conductance vessels have demonstrated that in endotoxemia iNOS overexpression contributes to the development of endothelial dysfunction, via a decreased activity of the endothelial isoform of NOS (eNOS) [8]. iNOS overexpression, therefore, could contribute to liver microvascular dysfunction during endotoxemia. Indeed, in a previous study we have shown that iNOS is markedly upregulated in the rat liver 24 h after LPS injection [3]. In addition, we have shown in a rat model of liver steatosis, which exhibits liver microvascular dysfunction, that iNOS inhibition improves liver endothelial function [9]. Altogether, this led us to hypothesize that iNOS upregulation could contribute to liver endothelial dysfunction and liver injury during endotoxemia. The present study was aimed at evaluating the role of iNOS upregulation in LPS-induced intrahepatic microvascular dysfunction and liver injury.

Materials and methods Animals and treatments Male Wistar rats, weighing 275–300 g, were caged in pairs in a 12/12 h light–dark cycle, temperature- and humidity-controlled environment. The animals were kept in environmentally controlled animal facilities at the Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS). All experiments were approved

Journal of Hepatology 2014 vol. 61 j 1321–1327

Research Article by the Laboratory Animal Care and Use Committee of the University of Barcelona and were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, 8th edition, 2011).

Experimental groups Rats were studied 24 h after the injection of LPS (5 mg/kg i.p.) or saline. The effects of LPS, as compared with saline injection, were evaluated in the presence or absence of the iNOS inhibitor 1400W (3 mg/kg, i.p.), given 3 and 23 h after the LPS/saline challenge. 1400W is a potent and selective iNOS inhibitor (>4000-fold selectivity for iNOS over eNOS and >130 fold selectivity for nNOS over eNOS) [10]. Previous in vivo data have shown that after a single administration, 1400W is highly biologically active in the colon, lung, liver, kidney, and heart [11].

Liver vascular studies After LPS/saline injection livers were isolated and perfused with Krebs buffer in a recirculation fashion with a total volume of 100 ml at a constant flow rate of 35 ml/min. An ultrasonic transit-time flow probe (model T201; Transonic Systems, Ithaca, NY) and a pressure transducer (Edwards Lifesciences, Irvine, CA) were placed on line, immediately ahead of the portal inlet cannula, to continuously monitor portal flow and perfusion pressure. Another pressure transducer was placed immediately after the thoracic vena cava outlet for the measurement of outflow pressure. The flow probe and the two pressure transducers were connected to a PowerLab (4SP) linked to a computer using the Powerlab software (chart version 5.5.4 for Windows, ADInstruments, Mountain View, LA). The average portal flow as well as inflow and outflow pressures were continuously sampled, recorded, and afterward blindly analysed under code. The perfused rat liver preparation was allowed to stabilize for 20 min before the studied substances were added. A normal gross appearance of the liver, a stable perfusion pressure, and a perfusate pH of 7.4 ± 0.1 were required during this period. Sinusoidal endothelial function was explored by testing the vasodilation of the liver circulation to increasing concentrations of acetylcholine (107, 106, 105 M) added to the system, after preconstruction with the alpha-adrenergic agonist methoxamine (104 M) following previously described techniques [12].

of the eNOS antibody together with 40 ll of washed protein A-coupled sepharose beads (4 °C, under continuous rotation) for precipitation. Resulting pellets were washed three times with PBS containing 01% Tween 20 and finally boiled with 80 ll of SDS–PAGE sample buffer. After centrifugation, supernatants were subjected to SDS–PAGE and Western blot analysis (40 ll supernatant/lane) for detection of caveolin-1 (BD Transduction Laboratories, Lexington, KY). Histopathology and immunohistochemistry Liver samples were fixed in 10% formalin, embedded in paraffin, sectioned (thickness of 2 lm), and slides were stained with haematoxylin and eosin (H&E). To specifically study leucocyte infiltration of the parenchyma immunostaining of paraffin-embedded liver sections was performed with anti-CD45 marker diluted 1:100 or, as a negative control, with phosphate-buffered saline in all groups of rats. The samples were photographed and analysed using a microscope (Zeiss, Jena, Germany) equipped with a digital camera. Oxidative stress In situ O 2 levels were evaluated with the oxidative fluorescent dye dihydroethidium (DHE; Molecular Probes) [15]. DHE specifically reacts with intracellular O 2 and is converted to the red fluorescent compound ethidium bromide, which then binds irreversibly to double-stranded DNA and appears as punctuate nuclear staining. Ethidium bromide is excited at 488 nm with an emission spectrum of 610 nm. Liver cryosections (10 lm) were incubated with DHE (10 lmol/L) in phosphate-buffered saline. Fluorescence images were obtained with a laser scanning confocal microscope (TCS-SL DMIRE2, Leica), and quantitative analysis was performed with Image J 1.33u software (National Institutes of Health). Serum biochemistry Blood samples, 1 ml, were obtained before liver perfusion for biochemistry. Buffer fluids from the liver perfusion studies were taken at the end of each experiment to analyse AST and ALT levels. All biochemical measurements were conducted with standard methods at our institution’s CORE lab.

Western blot analysis

Indocyanine green (ICG) clearance

At the end of the vascular study liver samples were obtained and immediately frozen in liquid nitrogen and kept at 80 °C until processed. Aliquots from each sample containing equal amounts of protein (40–100 lg) were run on an 8–15% SDS–polyacrylamide gel, and transferred to a nitrocellulose membrane. Equal loading was insured by Ponceau staining. The blots were subsequently blocked for 1 h with Tris-buffered saline and probed overnight at 4 °C with an antibody recognizing eNOS, iNOS (BD Transduction Laboratories, Lexington, KY), phosphorylated eNOS at Ser1176 (BD Transduction Laboratories, Lexington, KY), nitrotyrosine (Cayman Chemical Co, Ann Arbor, MI) and ET-1 (Thermo Scientific, Waltham, MA). This was followed by incubation with rabbit anti-mouse (1:10,000) or goat anti-rabbit (1:10,000) HRP-conjugated secondary antibodies (Stressgen, Glandford Ave, Victoria, BC, Canada) for 1 h at room temperature. Blots were revealed by chemiluminescence, and digital images were taken by the luminescent image analyser LAS-4000 (General Electric, Little Chalfont, Buckinghamshire, United Kingdom). Protein expression was determined by densitometric analysis using the Science Lab 2001, Multi Gauge V2.1 (Fuji Photo Film Gmbh, Düsseldorf, Germany). Quantitative densitometry values of iNOS, nitrotyrosine, ICAM-1, caspase-3 were normalized to GAPDH. The degree of eNOS phosphorylation at Ser1176 was calculated as the ratio between the densitometry readings of p-eNOS and eNOS blots. To evaluate the eNOS dimer-to-monomer ratio, low-voltage and low-temperature SDS-PAGE was run, using non-boiled tissue homogenates.

Rats were injected with ICG (0.5 mg/kg) through the femoral vein. Arterial blood samples (0.3 ml) were taken at baseline and 2, 4, and 15 min after the injection and centrifuged at 10,000g for 5 min. ICG absorbance was determined spectrophotometrically at a wavelength of 805 nm. Measured ICG absorbance was converted into the corresponding plasma concentration using a standard curve. ICG plasma disappearance rate was defined as the percentage decrease in ICG-plasma concentration per minute (%/min) [16,17].

eNOS/caveolin interaction Co-immunoprecipitation analyses were performed as previously described elsewhere [13,14]. In brief, for immunoprecipitation of eNOS, homogenates of rat liver were diluted. 40 lg protein were incubated overnight with 5 ll of the eNOS antibody. Then, samples were incubated for 180 min with 40 ll of washed protein G-coupled Sepharose beads (4 °C, under continuous rotation). Subsequently, beads were pelleted by centrifugation (14,000 rpm, 10 min, 4 °C). After preclearing and removal of beads, samples were incubated for 180 min with 5 ll

1322

Table 1. Biometric characteristics as well as rat glycaemia and alkaline phosphatase levels (ALP).

Control

1400W

Saline

LPS

Saline

LPS

Number of animals

8

8

7

9

Weight (gr)

319 ± 35

322 ± 26

334 ± 28

313 ± 41

Liver (% body weight)

2.7 ± 0.3

2.8 ± 0.2

2.9 ± 0.1

3.0 ± 0.4

Spleen (% body weight)

0.21 ± 0.04

0.28 ± 0.02* 0.23 ± 0.02

0.26 ± 0.03*

Glycemia (mg/dl) (plasma)

193 ± 16

97 ± 49*

162 ± 16

122 ± 10*

ALP (U/L) (plasma)

244 ± 51

678 ± 489*

228 ± 95

413 ± 105

Data are presented as mean ± SD. ⁄ p <0.05 vs. saline.

Journal of Hepatology 2014 vol. 61 j 1321–1327

JOURNAL OF HEPATOLOGY B

2.0

1.0 0.5 0.0 LPS

-6 -5 Acetylcholine (Log M)

D

% vasodilation

Results

Saline

Table 1 shows the baseline characteristics of the rats. All groups were comparable for body and liver weight. Those exposed to LPS showed a significant increase in spleen weight and alkaline phosphatase (ALP), and a decrease in plasma glycemia, not prevented by iNOS inhibition.

LPS

p-eNOS

LPS Saline

eNOS

10 p = 0.821

0

2.0

-10

p = 0.911

1.5

-20

iNOS upregulation contributes to liver microvascular dysfunction after LPS challenge

1.0

-30

0.5

-40 -50

+

Without 1400W

Without 1400W

C

-

Preacetylcholine -7

-6 -5 Acetylcholine (Log M) With 1400W

0.0 LPS

-

At 24 h after LPS/saline injection, iNOS expression was increased in livers of the LPS group as compared to the saline group (Supplementary Fig. 1). In keeping with our previous data [3], LPS induced a significant increase in baseline portal perfusion pressure (PPP; 6.3 ± 0.3 mmHg in the LPS group vs. 5.4 ± 0.4 mmHg in the saline group; p <0.001), indicating an increase in intrahepatic vascular resistance. In addition, LPS induced overt sinusoidal endothelial dysfunction, demonstrated by a decreased vasodilatory response to acetylcholine (p = 0.045) (Fig. 1A). This was associated with a reduction in liver eNOS phosphorylation at Ser1176 (p = 0.040), indicating decreased eNOS activation (Fig. 1B).

+

With 1400W

Fig. 1. Vasodilatory response to acetylcholine 24 h after LPS/saline administration and WB analysis for the p-eNOS/eNOS ratio. (A) LPS impaired the vasodilatory response to increasing doses of acetylcholine (intrahepatic endothelial dysfunction) (nsaline = 8 vs. nLPS = 8) and (B) decreased eNOS activation, as shown by a decreased p-eNOS/eNOS ratio (nsaline = 4 vs. nLPS = 5). (C) iNOS inhibition with 1400W prevented LPS-induced endothelial dysfunction (nsaline = 7 vs. nLPS = 9) and (D) restored eNOS activation (nsaline = 3 vs. nLPS = 3).

A

Control

LPS

LPS + 1400W

B

C

AST ALT

- LPS + LPS

100 H&E

ICG clearance [%/min]

80

CD45+

IU/L

60 40 20 0 +

-

20% reduction** 46% reduction

+

0W

-

20 18 16 14 12 10 8 6 4 2 0

40

LPS

p = 0.035

p = 0.001

*

W

Preacetylcholine -7

00

-40

W

ith

ou

t1

Without 1400W With 1400W

14

-30

20

p = 0.040

1.5

-20

ith

% vasodilation

eNOS

p = 0.045

-10

-50

Statistical analysis was performed using the SPSS 20.0 statistical package (IBM). Comparisons between groups were performed with the unpaired Student’s t test after confirming the assumptions of normality. Log-transformation was used to approximate normality when appropriate. Bootstrapping was used when normality was not achieved [18]. Dose response curves were analysed with repeated measures ANOVA, introducing LPS/saline exposure and treatment with 1400W/ saline as the between-subjects factors. Factorial analysis was used as appropriate to compare the changes induced by LPS amongst different treatment groups [19]. All data were reported as means ± SD. Differences were considered significant at a p value of less than 0.05.

p-eNOS

10 0

lin e LP S Sa lin e LP S

LPS Saline

W

20

Statistics

Sa

A

Fig. 2. Liver injury induced by LPS with or without the iNOS inhibitor 1400W. (A) Liver histology (H/E) and immunohistochemistry for CD-45. LPS induced perivenulitis (arrows) that was not prevented by 1400W. (B) AST and ALT levels in the perfusion fluid (without 1400W: nsaline = 4 vs. nLPS = 4; with 1400W: nsaline = 4 vs. nLPS = 4). LPS increased both AST and ALT leak, which was completely prevented by 1400W treatment (⁄change in AST and ALT in the 1400W group compared with the without 1400W group: p = 0.014 and p <0.001 respectively). (C) Indocyanine green (ICG) clearance. LPS administration induced a marked decrease in ICG clearance. This decrease was less pronounced in rats treated with 1400W, indicating that iNOS inhibition attenuated LPS-induced hepatocellular dysfunction (⁄⁄change in the 1400W group: p = 0.046 vs. change in the without 1400W group) (with 1400W nsaline = 2 vs. nLPS = 2; without 1400W nsaline = 3 vs. nLPS = 2).

Journal of Hepatology 2014 vol. 61 j 1321–1327

1323

Research Article 2.5

Hepatic ET-1 levels

lin LP e S

Sa

S LP

lin

e

p = 0.413

ET-1 GAPDH

2.0 1.5 1.0 0.5 -

+

LP

S

e lin Sa

S

lin

LP

Sa

S

lin

e

IP:eNOS

LP

B

e

0.0 LPS

Sa

iNOS upregulation contributes to hepatocellular damage induced by LPS

A Sa

In the group of rats treated with 1400W, LPS still induced an increase in PPP (6.8 ± 0.5 mmHg in the LPS group vs. 5.4 ± 0.4 mmHg in the saline group; p <0.001). In contrast, the development of sinusoidal endothelial dysfunction was markedly attenuated, to the point that response to acetylcholine was not significantly different between saline and LPS groups (Fig. 1C). In addition, iNOS inhibition prevented the decrease in eNOS phosphorylation induced by LPS (Fig. 1D). Therefore, these results suggest that iNOS upregulation contributes to LPS-induced intrahepatic endothelial dysfunction.

eNOS Caveolin-1

C

Without 1400W Saline

1324

Saline

LPS 260 kDa

eNOS

Dimer

130 kDa

Monomer

1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0

W 00 ith

14

40 t1 ou W

ith

- LPS + LPS

n.s.

0W

eNOS dimer/monomer ratio (a.u.)

Effects of iNOS inhibition on LPS-induced changes in caveolin-1 expression, eNOS uncoupling, and oxidative stress To better characterize the mechanisms mediating iNOS induced microvascular dysfunction in our model of endotoxemia we conducted additional molecular studies. Since in vitro data suggested that endothelin-1 (ET-1)-induced caveolin-1 (CAV1) overexpression participates in LPS-induced endothelial dysfunction [20,21], we first tested ET-1 levels and CAV1/eNOS interaction in liver lysates form groups treated with saline or LPS. We found no changes in intrahepatic levels of ET-1 after LPS, as compared to saline (Fig. 3A). In addition, co-immunoprecipitation did not show an increased interaction between eNOS and CAV1 after LPS (Fig. 3B). This suggests that ET-1-induced CAV1 expression is not a major contributor to liver endothelial dysfunction in our in vivo model of endotoxemia. It has been suggested that iNOS upregulation might reduce eNOS activity by competing for and limiting the availability of tetrahydrobiopterin (BH4), an essential cofactor both for iNOS and eNOS [8,22–24]. The principal marker of BH4 deficiency on eNOS is molecular uncoupling [25]. We studied in our model the ratio of coupled/uncoupled eNOS in livers exposed to LPS or saline, but no significant differences were found between the two groups (Fig. 3C), suggesting that a low availability of BH4 does not account for endothelial dysfunction in our model of endotoxemia. It has been previously shown that increased intrahepatic oxidative stress results in liver endothelial dysfunction [15], and we have previously shown that LPS increases liver oxidative stress [3]. Therefore, we investigated the effects of LPS and iNOS inhibition on liver oxidative stress by dihydroethidium (DHE)-staining (a surrogate of superoxide levels) and protein nitrotyrosination (a surrogate of nitrooxidative stress). As compared with saline injection, LPS caused liver oxidative stress, evidenced by increased liver DHE staining and increased liver protein nitrotyrosination (Fig. 4A). iNOS inhibition with 1400W blunted both the increase

LPS

With 1400 W

W

LPS induced liver inflammation, more marked in pericentral areas, with marked endothelitis at the central veins (Fig. 2A). In addition, LPS increased liver transaminases (Fig. 2B), indicating hepatocellular injury, and decreased hepatic ICG clearance, indicating hepatocellular dysfunction (Fig. 2C). iNOS inhibition did not significantly modify the degree of liver inflammation (Fig. 2A), but prevented hepatocellular injury. Indeed, no increase in AST and ALT levels was observed when LPS was given in the presence of 1400W (Fig. 2B), and the decrease in ICG clearance induced by LPS was attenuated by 1400W (Fig. 2C).

Fig. 3. Effects of LPS on liver ET-1 expression, caveolin-1/eNOS interaction and eNOS uncoupling. (A) ET-1 levels in liver lysates form groups treated with saline or LPS. LPS administration did not significantly modify hepatic ET-1 levels at 24 h (nsaline = 4 vs. nLPS = 5). (B) CAV1/eNOS interaction. eNOS was immunoprecipitated in liver lysates from rats treated with LPS or saline (nsaline = 3 vs. nLPS = 4). The immunoprecipitate was subjected to SDS–PAGE and Western blot analysis for eNOS and caveolin-1. Co-immunoprecipitation did not show an increased interaction between eNOS and CAV1 24 h after LPS administration. (C) eNOS uncoupling (in each experimental condition: nsaline = 3 vs. nLPS = 3). Non-boiled lysates were run on a low-voltage and low-temperature SDS-PAGE. The ratio of the eNOS dimer/monomer was calculated. There were no differences in the eNOS dimer to monomer ratio between the 4 study groups.

in liver DHE staining induced by LPS and the increase in protein nitrotyrosination (Fig. 4B). Altogether, these data suggest that iNOS induction, at least in part, might induce liver endothelial dysfunction and liver injury through an increase in liver nitrooxidative stress. Discussion In this study, we provide evidence showing that iNOS upregulation contributes to intrahepatic endothelial dysfunction (ED) and liver damage induced by LPS in a murine model of endotoxemia. Indeed, the pharmacological inhibition of iNOS activity after

Journal of Hepatology 2014 vol. 61 j 1321–1327

JOURNAL OF HEPATOLOGY e

+ LPS

25 kDa

3.5 3.0 p = 0.050 2.5 2.0 1.5 1.0 0.5 0.0 Without 1400W

S

GAPDH

Normalized intensity area

9 8 7 6 5 4 3 2 1 0 Without 1400W

- LPS + LPS

Normalized intensity area

Normalized intensity area

GAPDH

p = 0.014

25 kDa

NitroTyr

- LPS + LPS

9 8 7 6 5 4 3 2 1 0

p = 0.052

With 1400W

Normalized intensity area

NitroTyr

LP

LP

Sa

S

e

- LPS

lin

B

+ LPS

lin

- LPS

Sa

A

3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0

p=0.602

With 1400W

Fig. 4. Liver nitrotyrosine – oxidative stress. (A) LPS increased liver DHE staining and increased liver protein nitrotyrosination (DHE: nsaline = 6 vs. nLPS = 6; WB: nsaline = 3 vs. nLPS = 3). (B) iNOS inhibition with 1400W significantly blunted the increase in liver DHE staining induced by LPS, and attenuated the increase in protein nitrotyrosination (DHE: nsaline = 7 vs. nLPS = 7; WB: nsaline = 4 vs. nLPS = 5).

LPS injection markedly ameliorated endothelium-dependent vasodilation of the liver vasculature, blunted the increase in liver transaminases and improved the hepatic clearance of ICG. This occurred without any obvious effect on liver inflammation, and was associated with a marked attenuation in markers of nitrooxidative stress. Liver is a target organ in sepsis [1]. Up to 50% of patients with sepsis experience liver involvement [26]. In patients hospitalized for sepsis, not suffering from chronic liver disease, an early bilirubin increase at 48 h from admission is a marker of organ dysfunction and independently predicts survival [1]. Liver microcirculatory dysfunction has a strong impact on the viability of liver parenchymal and non-parenchymal cells [2], and this has been further emphasized by the demonstration of cytoprotective treatments improving liver microcirculation [3,27]. We have recently demonstrated that what occurs in conductance vessels and at the microcirculation of several organs, such as the heart, lung, brain and kidney [28] during endotoxemia endothelial dysfunction, also occurs at the hepatic microcirculation [3]. In this study, we additionally demonstrated that LPS induced liver iNOS expression, opening the possibility that iNOS could contribute to liver microvascular failure during endotoxemia. The upregulation of iNOS is one of the hallmarks of endothelitis [7]. Endothelial inflammation contributes to vascular dysfunction in several conditions, including atherosclerosis, hypertension, stroke, diabetes and septic shock [7,29–32]. In the present study, we expanded this list of conditions to liver microvascular dysfunction during endotoxemia. Our study demonstrates that iNOS inhibition prevents microvascular dysfunction and may improve hepatocyte perfusion and metabolism in our rat model of endotoxemia. This is in keeping with results obtained in resistance and conduit vessels, in which iNOS induction has been shown to contribute to endothelial dysfunction induced by LPS. Indeed, iNOS knockout mice (iNOS/) are completely protected from LPSinduced endothelial dysfunction in the peripheral circulation [33]. Several mechanisms have been proposed to establish a link between endotoxemia and endothelial dysfunction [8,20,34,35], but most were documented only in cell culture studies. In the present study we wanted to further assess, in a complex in vivo

system, if some of these mechanisms could account for LPSinduced liver endothelial dysfunction. First, we addressed ET-1eNOS interaction. Kamoun et al. [20] recently suggested that LPS does suppress ET-1-induced eNOS activation via an upregulation of caveolin-1 (which interacts with eNOS, inhibiting eNOS activity [36,37]). In addition, these authors demonstrated whole-liver caveolin upregulation 6 h after LPS injection. In the present study we could not demonstrate an increased interaction between caveolin-1 and eNOS 24 h after LPS. Though we performed our studies in whole liver lysates, we first immunoprecipitated eNOS, which is negligibly expressed outside the liver endothelium [38,39], and then assessed caveolin-1 expression in the precipitate. In our view, this excludes reasonably an increased eNOS-CAV1 interaction at the liver endothelium 24 h after LPS challenge. We then addressed whether iNOS upregulation could induce eNOS uncoupling. A previous study in isolated peripheral vessels suggested that vascular iNOS upregulation competes with eNOS for BH4 [8]. This cofactor is essential for eNOS dimerization, preventing eNOS uncoupling and malfunction [23,24,40]. Again, our results do not suggest that this is a major mechanisms acting in the liver during endotoxemia, since in the present model we could not demonstrate increased eNOS uncoupling in liver lysates from rats exposed to LPS. Finally, our findings suggest that iNOS inhibition attenuates the increase in oxidative stress induced by endotoxemia, as demonstrated by both a decrease in superoxide bioavailability (assessed by DHE staining) and protein nitrotyrosination. On one hand, it is well established that increased oxidative stress contributes to liver vascular dysfunction [15,41], and therefore this could be a major mechanisms mediating the liver protective effects of early iNOS inhibition in endotoxemia. On the other hand an improvement in endothelial function and liver perfusion in itself could lead to decreased oxidative stress within the liver [15], making it difficult to dissect whether the antioxidant effects of iNOS inhibition are the cause or the consequence of the improvement in liver endothelial function. This limitation is one of the inherent drawbacks of in vivo studies. While providing a valuable synthesis of the several interactions that occur in complex systems, they can only offer limited mechanistic detail.

Journal of Hepatology 2014 vol. 61 j 1321–1327

1325

Research Article Another question raised by our data is whether cell-specific iNOS upregulation determines LPS-induced microvascular dysfunction. Previous studies have shown that LPS upregulates iNOS in hepatocytes, Kupffer cells, infiltrating inflammatory cells, and liver sinusoidal endothelial cells [42–44]. A definite answer to this question would require the comparison of the effects of LPS/saline in different genetically modified animals, including iNOS/ mice, endothelial specific iNOS/ mice and irradiated iNOS/ mice reconstituted with wild type bone marrow cells [43], to dissect the specific role of iNOS upregulation in parenchymal, endothelial and bone marrow derived cells. Our findings might be of special relevance for cirrhosis, in which endotoxemia is a prominent feature [45]. Indeed, previous data showed iNOS induction in the mesenteric circulation of cirrhotic rats [46–48] especially in those with ascites [46], suggesting that a highly selective iNOS inhibitor could potentially target vascular abnormalities both in the liver and systemic circulation in advanced cirrhosis. In conclusion, our study demonstrates in a murine model that iNOS upregulation contributes to liver microvascular dysfunction in endotoxemia. This suggests that this mechanism deserves further exploration in studies addressing liver protection in the context of severe acute bacterial infection, or clinical situations in which liver dysfunction is a prominent feature of sepsis, such as cirrhosis.

[2]

[3]

[4]

[5] [6]

[7]

[8]

[9]

[10] [11]

[12]

Financial support [13]

This study was supported by a grant from the Instituto de Salud Carlos III (FIS PI11/00883 to JGA and PI13/00341 to JB), and cofinanced by FEDER funds (EU, ‘‘Una manera de hacer Europa’’). Part of this work was carried out at the Esther Koplowitz Centre, Barcelona. Ciberehd is funded by the Instituto de Salud Carlos III.

Conflict of interest

[14]

[15]

[16]

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.

[17]

[18]

Authors’ contributions [19]

Study concept and design: JGA, JB, VLM. Acquisition of data: VLM, MP, RVA. Drafting of the manuscript: VLM, MP. Critical revision of the manuscript: JGA, JB, JGP, MP, VLM. Statistical analysis: VLM, MP, JGA. Obtained funding: JGA, JB. Study supervision: JGA, JB.

[20]

[21]

Supplementary data [22]

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jhep.2014.07. 014.

[23]

[24]

References [25] [1] Kramer L, Jordan B, Druml W, Bauer P, Metnitz PG. Austrian Epidemiologic Study on Intensive Care ASG. Incidence and prognosis of early hepatic

1326

dysfunction in critically ill patients – A prospective multicenter study. Crit Care Med 2007;35:1099–1104. Vollmar B, Menger MD. The hepatic microcirculation: mechanistic contributions and therapeutic targets in liver injury and repair. Physiol Rev 2009;89:1269–1339. La Mura V, Pasarin M, Meireles CZ, Miquel R, Rodriguez-Vilarrupla A, Hide D, et al. Effects of simvastatin administration on rodents with lipopolysaccharide-induced liver microvascular dysfunction. Hepatology 2013;57: 1172–1181. Dangas G, Smith DA, Unger AH, Shao JH, Meraj P, Fier C, et al. Pravastatin: an antithrombotic effect independent of the cholesterol-lowering effect. Thromb Haemost 2000;83:688–692. Lefer DJ. Statins as potent antiinflammatory drugs. Circulation 2002;106: 2041–2042. Bates K, Ruggeroli CE, Goldman S, Gaballa MA. Simvastatin restores endothelial NO-mediated vasorelaxation in large arteries after myocardial infarction. Am J Physiol 2002;283:H768–H775. Borutaite V, Moncada S, Brown GC. Nitric oxide from inducible nitric oxide synthase sensitizes the inflamed aorta to hypoxic damage via respiratory inhibition. Shock 2005;23:319–323. Gunnett CA, Lund DD, McDowell AK, Faraci FM, Heistad DD. Mechanisms of inducible nitric oxide synthase-mediated vascular dysfunction. Arterioscler Thromb Vasc Biol 2005;25:1617–1622. Pasarin M, Abraldes JG, Rodriguez-Vilarrupla A, La Mura V, Garcia-Pagan JC, Bosch J. Insulin resistance and liver microcirculation in a rat model of early NAFLD. J Hepatol 2011;55:1095–1102. Vallance P, Leiper J. Blocking NO synthesis: how, where and why? Nat Rev Drug Discov 2002;1:939–950. Garvey EP, Oplinger JA, Furfine ES, Kiff RJ, Laszlo F, Whittle BJ, et al. 1400W is a slow, tight binding, and highly selective inhibitor of inducible nitric-oxide synthase in vitro and in vivo. J Biol Chem 1997;272:4959–4963. Abraldes JG, Rodriguez-Vilarrupla A, Graupera M, Zafra C, Garcia-Caldero H, Garcia-Pagan JC, et al. Simvastatin treatment improves liver sinusoidal endothelial dysfunction in CCl4 cirrhotic rats. J Hepatol 2007;46:1040–1046. Karaa A, Kamoun WS, Clemens MG. Oxidative stress disrupts nitric oxide synthase activation in liver endothelial cells. Free Radic Biol Med 2005;39:1320–1331. Ashburn JH, Baveja R, Kresge N, Korneszczuk K, Keller S, Karaa A, et al. Remote trauma sensitizes hepatic microcirculation to endothelin via caveolin inhibition of eNOS activity. Shock 2004;22:120–130. Gracia-Sancho J, Lavina B, Rodriguez-Vilarrupla A, Garcia-Caldero H, Fernandez M, Bosch J, et al. Increased oxidative stress in cirrhotic rat livers: a potential mechanism contributing to reduced nitric oxide bioavailability. Hepatology 2008;47:1248–1256. Kawai T, Yokoyama Y, Nagino M, Kitagawa T, Nimura Y. Is there any effect of renal failure on the hepatic regeneration capacity following partial hepatectomy in rats? Biochem Biophys Res Commun 2007;352:311–316. Raddatz A, Kubulus D, Winning J, Bauer I, Pradarutti S, Wolf B, et al. Dobutamine improves liver function after hemorrhagic shock through induction of heme oxygenase-1. Am J Respir Crit Care Med 2006;174: 198–207. Calmettes G, Drummond GB, Vowler SL. Making do with what we have: use your bootstraps. J Physiol 2012;590:3403–3406. Drummond GB, Vowler SL. Analysis of variance: variably complex. J Physiol 2012;590:1303–1306. Kamoun WS, Karaa A, Kresge N, Merkel SM, Korneszczuk K, Clemens MG. LPS inhibits endothelin-1-induced endothelial NOS activation in hepatic sinusoidal cells through a negative feedback involving caveolin-1. Hepatology 2006;43:182–190. Kwok W, Lee SH, Culberson C, Korneszczuk K, Clemens MG. Caveolin-1 mediates endotoxin inhibition of endothelin-1-induced endothelial nitric oxide synthase activity in liver sinusoidal endothelial cells. Am J Physiol Gastrointest Liver Physiol 2009;297:G930–G939. Andrew PJ, Mayer B. Enzymatic function of nitric oxide synthases. Cardiovasc Res 1999;43:521–531. Kinoshita H, Milstien S, Wambi C, Katusic ZS. Inhibition of tetrahydrobiopterin biosynthesis impairs endothelium-dependent relaxations in canine basilar artery. Am J Physiol 1997;273:H718–H724. Matei V, Rodriguez-Vilarrupla A, Deulofeu R, Colomer D, Fernandez M, Bosch J, et al. The eNOS cofactor tetrahydrobiopterin improves endothelial dysfunction in livers of rats with CCl4 cirrhosis. Hepatology 2006;44:44–52. Alp NJ, Channon KM. Regulation of endothelial nitric oxide synthase by tetrahydrobiopterin in vascular disease. Arterioscler Thromb Vasc Biol 2004;24:413–420.

Journal of Hepatology 2014 vol. 61 j 1321–1327

JOURNAL OF HEPATOLOGY [26] Flum DR, Dellinger EP, Cheadle A, Chan L, Koepsell T. Intraoperative cholangiography and risk of common bile duct injury during cholecystectomy. JAMA 2003;289:1639–1644. [27] Gracia-Sancho J, Garcia-Caldero H, Hide D, Marrone G, Guixe-Muntet S, Peralta C, et al. Simvastatin maintains function and viability of steatotic rat livers procured for transplantation. J Hepatol 2013;58:1140–1146. [28] Vallet B. Bench-to-bedside review: endothelial cell dysfunction in severe sepsis: a role in organ dysfunction? Critical care 2003;7:130–138. [29] Bardell AL, MacLeod KM. Evidence for inducible nitric-oxide synthase expression and activity in vascular smooth muscle of streptozotocindiabetic rats. J Pharmacol Exp Ther 2001;296:252–259. [30] Esaki T, Hayashi T, Muto E, Kano H, Kumar TN, Asai Y, et al. Expression of inducible nitric oxide synthase and Fas/Fas ligand correlates with the incidence of apoptotic cell death in atheromatous plaques of human coronary arteries. Nitric Oxide 2000;4:561–571. [31] Kibbe M, Billiar T, Tzeng E. Inducible nitric oxide synthase and vascular injury. Cardiovasc Res 1999;43:650–657. [32] Llorens S, Nava E. Cardiovascular diseases and the nitric oxide pathway. Curr Vasc Pharmacol 2003;1:335–346. [33] Chauhan SD, Seggara G, Vo PA, Macallister RJ, Hobbs AJ, Ahluwalia A. Protection against lipopolysaccharide-induced endothelial dysfunction in resistance and conduit vasculature of iNOS knockout mice. FASEB J 2003;17:773–775. [34] Cuzzocrea S, Mazzon E, Di Paola R, Esposito E, Macarthur H, Matuschak GM, et al. A role for nitric oxide-mediated peroxynitrite formation in a model of endotoxin-induced shock. J Pharmacol Exp Ther 2006;319:73–81. [35] Asakura H, Asamura R, Ontachi Y, Hayashi T, Yamazaki M, Morishita E, et al. Selective inducible nitric oxide synthase inhibition attenuates organ dysfunction and elevated endothelin levels in LPS-induced DIC model rats. J Thromb Haemost 2005;3:1050–1055. [36] Shah V, Toruner M, Haddad F, Cadelina G, Papapetropoulos A, Choo K, et al. Impaired endothelial nitric oxide synthase activity associated with enhanced caveolin binding in experimental cirrhosis in the Rat. Gastroenterology 1999;117:1222–1228. [37] Fleming I, Busse R. Molecular mechanisms involved in the regulation of the endothelial nitric oxide synthase. Am J Physiol Regul Integr Comp Physiol 2003;284:R1–12.

[38] Iwakiri Y. Endothelial dysfunction in the regulation of cirrhosis and portal hypertension. Liver Int 2012;32:199–213. [39] Pasarin M, La Mura V, Gracia-Sancho J, Garcia-Caldero H, RodriguezVilarrupla A, Garcia-Pagan JC, et al. Sinusoidal endothelial dysfunction precedes inflammation and fibrosis in a model of NAFLD. PLoS one 2012;7:e32785. [40] Matei V, Rodriguez-Vilarrupla A, Deulofeu R, Garcia-Caldero H, Fernandez M, Bosch J, et al. Three-day tetrahydrobiopterin therapy increases in vivo hepatic NOS activity and reduces portal pressure in CCl4 cirrhotic rats. J Hepatol 2008;49:192–197. [41] Lavina B, Gracia-Sancho J, Rodriguez-Vilarrupla A, Chu Y, Heistad DD, Bosch J, et al. Superoxide dismutase gene transfer reduces portal pressure in CCl4 cirrhotic rats with portal hypertension. Gut 2009;58:118–125. [42] Vos TA, Van Goor H, Tuyt L, Jager-Krikken A, Leuvenink R, Kuipers F, et al. Expression of inducible nitric oxide synthase in endotoxemic rat hepatocytes is dependent on the cellular glutathione status. Hepatology 1999;29:421–426. [43] Bultinck J, Sips P, Vakaet L, Brouckaert P, Cauwels A. Systemic NO production during (septic) shock depends on parenchymal and not on hematopoietic cells: in vivo iNOS expression pattern in (septic) shock. FASEB J 2006;20:2363–2365. [44] Rockey DC, Chung JJ. Regulation of inducible nitric oxide synthase and nitric oxide during hepatic injury and fibrogenesis. Am J Physiol 1997;273: G124–G130. [45] Wiest R, Lawson M, Geuking M. Pathological bacterial translocation in liver cirrhosis. J Hepatol 2014;60:197–209. [46] Angeli P, Fernandez-Varo G, Dalla Libera V, Fasolato S, Galioto A, Arroyo V, et al. The role of nitric oxide in the pathogenesis of systemic and splanchnic vasodilation in cirrhotic rats before and after the onset of ascites. Liver Int 2005;25:429–437. [47] Bhimani EK, Serracino-Inglott F, Sarela AI, Batten JJ, Mathie RT. Hepatic and mesenteric nitric oxide synthase expression in a rat model of CCl(4)-induced cirrhosis. J Surg Res 2003;113:172–178. [48] Malyshev E, Tazi KA, Moreau R, Lebrec D. Discrepant effects of inducible nitric oxide synthase modulation on systemic and splanchnic endothelial nitric oxide synthase activity and expression in cirrhotic rats. J Gastroenterol Hepatol 2007;22:2195–2201.

Journal of Hepatology 2014 vol. 61 j 1321–1327

1327