Short Title: Effects of Norepinephrine In Hepatic Stellate Cells

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Norepinephrine Induces Calcium Spikes And Proinflammatory

Actions In Human Hepatic Stellate Cells.

Short Title: Effects of Norepinephrine In Hepatic Stellate Cells

Pau Sancho-Bru1, Ramón Bataller1, Jordi Colmenero1, Xavier Gasull2, Montserrat

Moreno1, Vicente Arroyo1, David A. Brenner3, Pere Ginès1.

1Liver Unit, Institut Clínic de Malalties Digestives i Metabòliques, Hospital Clínic,

2Laboratory of Neurophysiology, University of Barcelona School of Medicine, Institut

d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Catalonia,

Spain. 3Department of Medicine, Columbia University College of Physicians and

Surgeons, New York, NY.

Keywords: portal hypertension, collagen, inflammation, catecholamines, liver fibrosis.

Corresponding author:

Ramón Bataller, M.D.

Liver Unit, Hospital Clínic

Villarroel 170

08036-BARCELONA

Spain

Phone 34-93-227.54.10

E-mail: [email protected]

of 34Articles in PresS. Am J Physiol Gastrointest Liver Physiol (June 15, 2006). doi:10.1152/ajpgi.00537.2005

Copyright © 2006 by the American Physiological Society.

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ABSTRACT

Background and aims: Catecholamines participate in the pathogenesis of portal

hypertension and liver fibrosis through α1 adrenoceptors. However, the underlying

cellular and molecular mechanisms are largely unknown. Here, we investigate the

effects of norepinephrine (NE) on human hepatic stellate cells (HSC), which exert

vasoactive, inflammatory and fibrogenic actions in the injured liver.

Methods: Adrenoceptor expression was assessed in human HSC by RT-PCR and

immunocytochemistry. Intracellular calcium concentration ([Ca2+]i) was studied in fura-

2 loaded cells. Cell contraction was studied by assessing wrinkle formation and myosin

light chain II (MLC-II) phosphorylation. Cell proliferation and collagen-α1(I)

expression were assessed by 3H-thymidine incorporation and quantitative PCR,

respectively. NFκB activation was assessed by luciferase reporter gene and p65 nuclear

translocation. Chemokine secretion was assessed by ELISA.

Results: Normal human livers expressed α1A adrenoceptors, which were markedly up-

regulated in livers with advanced fibrosis. Activated human HSC expressed α1A

adrenoceptors. Norepinephrine induced multiple rapid [Ca2+]i oscillations (Ca2+ spikes).

Prazosin (α1 blocker) completely prevented NE-induced Ca2+ spikes, while propranolol

(nonspecific β blocker) partially attenuated this effect. Norepinephrine caused

phosphorylation of MLC-II, and cell contraction. In constrast, NE did not affect cell

proliferation nor collagen α1(I) expression. Importantly, NE stimulated the secretion of

inflammatory chemokines (RANTES and interleukin-8) in a dose-dependent manner.

Prazosin blocked NE-induced chemokine secretion. Norepinephrine stimulated NFκB

activation. BAY11-7082, a specific NFκB inhibitor, blocked NE-induced chemokine

secretion.

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Conclusions: NE stimulates NFκB and induces cell contraction and proinflammatory

effects in human HSC. Catecholamines may participate in the pathogenesis of portal

hypertension and liver fibrosis by targeting HSC.

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INTRODUCTION

Catecholamines regulate a number of physiological functions in the liver. The

normal human liver contains abundant sympathetic innervation and circulating

catecholamines regulate hepatic vascular tone as well as hepatic metabolic functions

(10; 13; 43). In patients with cirrhosis, catecholamines may participate in the

pathogenesis of portal hypertension. In these patients, elevated serum catecholamines

are believed to contribute to the increased intrahepatic vascular resistance to blood flow

(4; 16). Moreover, α and β adrenoceptor blockers markedly decrease portal pressure.

The effect of nonselective β blockers is mainly mediated by a reduction in portal venous

blood flow, while the beneficial effects of α1 receptor blockers is basically due to a

reduction in intrahepatic resistance (3; 13). In rats with experimentally-induced

cirrhosis, α1 adrenoceptor agonists increase intrahepatic vascular resistance (17). We

hypothesize that α1 adrenoceptors induce vasoactive effects in contractile -

myofibroblastic- hepatic cell types, which might mediate changes in vascular tone in

advanced liver diseases.

Besides its role in the regulation of vascular tone, catecholamines seem to be

involved in the wound healing response to injury (11; 15). Locally released

catecholamines regulate key biological features of the tissue repair process, including

myofibroblast proliferation and collagen secretion (1; 2). Therefore, catecholamines are

currently viewed as a system that participates in local response to chronic injury. Recent

evidence suggests that catecholamines may participate in the pathogenesis of liver

fibrogenesis (29). Experimentally-induced liver fibrosis is enhanced in spontaneously

hypertensive rats, which have elevated serum levels of catecholamines (21). Moreover,

both chemical sympathectomy and treatment with prazosin, a α1 adrenoceptor blocker,

attenuate experimentally-induced liver fibrosis in rats (15; 30). Finally, genetic ablation

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of the sympathetic nervous system (SNS) reduces liver fibrosis in mice (32). Moreover,

it has been shown that rodent HSC express some adrenoceptor subtypes, and that

stimulation with NE induces proliferation and collagen gene expression (32; 33).

The cell type mediating the beneficial effect of α1 adrenoceptor blockers on

portal hypertension and liver fibrosis are unknown. Hepatic stellate cells (HSC) are

potential targets for the vasoactive actions of catecholamines in the liver. Contraction of

HSC in response to vasoconstrictors (i.e. endothelin-1 and angiotensin II) are believed

to contribute to the increased resistance to blood flow in advanced chronic liver diseases

(6; 35-37). Moreover, activated HSC secrete large amounts of collagen I as well as

inflammatory mediators in chronic liver diseases, regulating fibrogenesis and

inflammation (27). We hypothesize that NE, a major catecholamine, induces vasoactive

effects on activated HSC. Besides this action, we explore whether NE also regulates the

fibrogenic and proinflammatory actions in these cells.

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MATERIALS AND METHODS

Human liver specimens and HSC isolation. Human cirrhotic livers were

obtained from liver explants at the time of liver transplantation from patients with end-

stage liver disease due to hepatic C virus infection (N=5). Human control livers were

obtained from resections of liver metastasis of colon cancer (N=5). Human HSC were

isolated from fragments of normal livers as described in detail previously (6).

Experiments were performed with HSC activated in culture (after the second serial

passage). A subset of experiments were performed in activated non passaged HSC. The

immunocytochemistry studies were performed also in HSC freshly isolated from normal

human livers (quiescent phenotype). In all cell cultures, no staining was found for

CD45, factor VIII related-antigens, and cam 5.2 (Dako, Glostrup, Denmark), indicating

the absence of mono/macrophagic, endothelial, and epithelial cells. Cells were cultured

in standard conditions in Iscove’s Modified Dulbecco’s Medium (IMDM,

BioWhittaker, Verviers, Belgium) containing 15% fetal bovine serum. Cells were serum

starved for at least 12 hours before the experiments. The protocol was approved by the

Investigational Review Board of the Hospital Clínic of Barcelona.

Immunocytochemistry studies. Cultured HSC were fixed in methanol at -20ºC

for 10 min, blocked in PBS containing 0.1% BSA for 30 min, and incubated with anti-

p65 or anti-α1A-adrenoceptor for 1 hour (Santa Cruz Biotechnology. Santa Cruz. CA).

Cells were incubated with Cy3 or fluorescein-labeled secondary antibody for 1 hour. An

isotype-matched antibody was used as a negative control. Cells were analyzed using a

Leica TCS SL laser scanning confocal spectral microscope (Leica Microsystems

Heidelberg GmbH, Manheim, Germany) at a wavelength of 500-535 nm.

Measurement of intracellular Ca2+ concentration ([Ca++]i). Changes in [Ca2+]i

were measured in Fura-2 (Calbiochem, San Diego, CA) loaded cells using an inverted

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epifluorescence microscope as described in detail previously (6). A representative

experiment was performed in Fura-4 loaded cells in a confocal spectral microscope.

Cells were tested with NE, prazosin and propranolol (Sigma Chemical Co, St Luis, MI).

To study Ca2+ source, experiments were performed with and without extracellular

calcium in selected groups. To deplete intracellular Ca2+ stores, cells were incubated

with thapsigargin (Sigma) for 20 minutes before NE stimulation. Cells were considered

as responders when [Ca2+]i increased more than 50% above the resting value.

Assessment of cellular contraction: The contractile responses of cultured HSC

to NE were evaluated by wrinkle formation (19; 24). A thin layer of the polydimethyl

siloxane (12.500 cP; Sigma) was spread on microscope cover slips. The silicone fluid

layer was briefly heated on a low flame from a Bunsen burner to promote cross-linking

of the silicone fluid surface while the overlying non-cross-linked fluid served as a

lubricant and allowed the surface to move independently of the cover slip. Cells plated

on the silicone rubber membrane showed basal wrinkles after 2-3 days in culture. Cells

were photographed every 5 min on an Olympus IX70 inverted phase-contrast

microscope with a digital coupled device camera (Hamamatsu, Shizuoka, Japan) and

stimulated with the agonists to assess cellular contraction by wrinkle formation. Cell

contraction was considered when there was a significant increase in the number of

wrinkles or an increase in the length of the preexisting ones.

Gene expression assays. RNA was isolated from activated HSC using Trizol

(Life Technologies Inc., Rockville, MD). PCR primer pairs were designed to amplify

GAPDH, and adrenoceptors α1A, α1B, α2A, α2B, β1, and β2 (Table 1). Quantitative PCR

was performed with pre-designed Assays-on-Demand TaqMan probes and primer pairs

for collagen α1(I) and ribosome subunit 18s. Information on these Assay-on-Demand is

available at: http://myscience.appliedbiosystems.com/cdsEntry/Form/gene_expression

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_keyword.jsp. TaqMan reactions were carried out in duplicate on an ABI PRISM 7900

machine (Applied Biosystems, Foster City, CA).

Cell proliferation assay. DNA synthesis was estimated by methyl-3H thymidine

(Amersham Biosciences, Buckinghamshire, UK) incorporation, as described in detail

previously (7). Cells were serum starved for 24h, stimulated for 18h with agonists and

then pulsed for 6h with 1µCi/ml methyl-3H thymidine. Results are expressed as fold-

stimulation compared to cells incubated with buffer.

Determination of chemokine secretion. HSC were cultured in 6-well plates at a

density of 4x105 cells/well for 24 h. Medium was removed and cells incubated in

serum-free medium for 24 h in the presence of agonists. Supernatants were collected

and a sandwich ELISA for human interleukin-8 (IL-8) (BLK Diagnostics, Barcelona,

Spain) and RANTES (R&D Systems, Minneapolis, MN) was performed. Cells were

preincubated with SP600125, PD231445 (Sigma), and BAY11-7082 (Calbiochem,

Darmstadt, Germany) in indicated experiments. Results are expressed as fold-increase

chemokine secretion compared to treated with buffer cells.

Recombinant Adenoviral Infection. Recombinant adenoviral vectors expressing

either a luciferase reporter gene driven by NF-κB transcriptional activation (Ad5NF-

κBLuc) (39) a dominant negative mutant form (S32A/S36A) of IκB (Ad5IκB) (23), or a

control green fluorescent protein (Ad5GFP) (28) were used. HSCs were infected with

Ad5NF-κBLuc (MOI 500) and/or Ad5IκB (MOI 1,000), Ad5GFP (MOI 1,000) for 12

hours in DMEM containing 0.5% FCS. After infection, the medium was replaced with

medium containing 0.5% FCS, and the culture was continued for additional 8 hours

before performing the individual experiments.

NFκB responsive luciferase assay. HSC were infected with Ad5NF-κBLuc for

12 hours. Medium was replaced and cells stimulated with agonists for 8 hours. NFκB-

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mediated transcriptional induction was assessed by the luciferase assay system (BD

Pharmigen, San Diego, CA). Luciferase activity (relative light units) was normalized to

the protein concentration.

Electrophoretic mobility shift assay (EMSA). Cell nuclear proteins were

extracted as described previously (8). Eight µg of nuclear proteins were incubated with

100 pg of a 32P-labeled probe containing the activating protein-1 (AP-1) consensus site

(5’-GTAAAGCATGAGTCAGACACCTC-3’) in buffer containing 10 mM HEPES pH

7.8, 2 mM MgCl2, 50 mM KCl, 1 mM DTT, 0.1 mM EDTA and 20% glycerol in the

presence of single stranded oligonucleotide (25 µg/ml) and poly dI/dC (25 µg/ml) for 20

minutes at room temperature. For competition assay, one sample was incubated with 10

ng unlabelled probe.

Western blotting. Whole cell extracts were obtained in Triton lyses buffer

containing protease and phosphatase inhibitors. Twenty-five µg were loaded onto 10%

or 15% SDS-acrylamide gels and blotted onto nitrocellulose membranes. Membranes

were then incubated with antibodies against SAPK/JNK, phospho-SAPK/JNK, p44/42

MAP Kinase (ERK), phospho-ERK, phospho myosin light chain-II (MLC-II) (Cell

Signaling, Beverly, MA) and α1A-adrenoceptor (Santa Cruz Biotechnology). After

extensive washing, membranes were incubated with blocking buffer containing

horseradish-peroxidase conjugated secondary antibody. Proteins were detected by ECL

chemoluminescence (Amersham).

Data analysis. Data presented herein are representative of at least three

experiments. Results are expressed as mean ± SD. Statistical analysis was performed by

Student's t test and analysis of variance.

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RESULTS

α1A adrenergic receptors are expressed in human HSC and are up-regulated

in human fibrotic livers. We first assessed the expression of adrenoceptors in human

HSC by RT-PCR. Human cultured HSC express α1A, α2B, and β2 adrenoceptors (Figure

1A). In contrast, α1B, α2A and β1 adrenoceptors were not expressed in HSC but were

detected in RNA obtained from total human liver. Because α1 receptors mediate key

biological effects of the SNS in fibrogenic cell types (15; 38), we further investigated

their expression in HSC as well as in normal and fibrotic human liver specimens. Both

quiescent and culture-activated HSC contain α1A receptors, as assessed by

immunocytochemistry (Figure 1B, 1C and 1D). Moreover, western blotting analysis of

whole liver extracts revealed that normal human livers express α1A receptors, which are

markedly up-regulated in livers with advanced fibrosis (Figure 2). These results suggest

that α1A receptors are up-regulated during liver fibrogenesis and that HSC are potential

targets for adrenergic agonists and antagonist.

Norepinephrine induces Ca2+ spikes and cell contraction in human HSC. We

next stimulated HSC with NE, a major neurotransmitter of the SNS. Stimulation of cells

with NE (10 µM) increased [Ca2+]i in Fura-2 loaded HSC (table 2 and figure 3A and

3D). The typical pattern was characterized by an abrupt increase of [Ca2+]i followed by

an oscillatory response in 57% of the cells (Ca2+ spikes). Ten µM NE induced a

significant increase of [Ca2+]i in 84% of the cells and a mean Ca2+ peak of 317±138 nM.

Cell stimulation with 1 and 0.1 µM NE also induced a marked [Ca2+]i increase (not

shown). Removal of Ca2+ from the extracellular solution did not affect NE-induced Ca2+

response. Moreover, cell preincubation with thapsigargin, a cell-permeable SERCA

pump inhibitor that depletes Ca2+ intracellular stores, completely abolished NE-induced

Ca2+ response (data not shown). These results indicate NE mainly releases Ca2+ from

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intracellular stores. Prazosin completely prevented NE-induced [Ca2+]i increase in all

cells (Table 2 and figure 3B). However, preincubation of cells with propranolol only

attenuated NE-induced [Ca2+]i peak and the percentage of cells showing an oscillatory

response (Figure 3C). Similar results were obtained with non-passaged cells activated in

culture (data not shown) We next explored whether NE induces cell contraction. NE

induced MLC-II phosphorylation after 5 min, which is a known prerequisite for cell

contraction (20; 44) (Figure 3E). Preincubation with 10µM of BAPTA-AM did not

significantly modify MLC-II phosphorylation, suggesting that it could be calcium-

independent (data not shown). Moreover, we found that stimulation with NE induced

cell contraction, as assessed by wrinkle formation (Figure 3F). Taken together, these

results indicate that NE induces cell contraction and may be involved in the vasoactive

effects of HSC.

Norepinephrine does not stimulate proliferation of human HSC. We

investigated whether NE regulates human HSC growth. Different concentrations of NE

(10nM - 10µM) were tested. Incubation with NE did not affect DNA synthesis, as

assessed by 3H-thymidine incorporation (Figure 4A). Stimulation with phenilephrine or

terbutaline (an α and β receptor agonist, respectively) did not affect cell proliferation

(data not shown). To elucidate whether NE regulates agonist-induced cell proliferation,

HSC were pretreated with NE (10 µM) and then challenged with platelet derived growth

factor-BB (PDGF-BB) (20 ng/ml), a potent mitogen for these cells. NE pretreatment did

not affect PDGF-induced mitogenic effect. These results indicate that NE is not

mitogenic for human HSC.

Effect of norepinephrine on gene expression of collagen α1(I) in human HSC.

We next investigated whether NE regulates the expression of collagen α1(I), the main

extracellular protein found in fibrotic human livers. Stimulation of cells with NE (10

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µM) did not affect collagen expression, as assessed by quantitative PCR (Figure 4B).

These results suggest that NE does not directly regulate collagen synthesis in human

activated HSC.

Norepinephrine stimulates chemokine production by human HSC. We next

investigated whether NE regulates the inflammatory actions of HSC by stimulating

chemokine secretion. Norepineprhine (10 µM) induced a significant increase in

secretion of IL-8 and RANTES to the culture media, which are chemokines potentially

involved in liver fibrogenesis (34; 40) (Figure 5A). Preincubation of cells with prazosin

markedly reduced both IL-8 and RANTES secretion. Propranolol also attenuated NE-

induced chemokine production, yet only RANTES secretion was significantly reduced.

These results suggest that the inflammatory actions of NE on human HSC could be

mediated by both α1 and β adrenoceptors.

Effect of NE on intracellular signaling pathways in human HSC. We finally

explored the effect of NE on intracellular pathways known to regulate the inflammatory

actions in human HSC (8; 41). These pathways include mitogen-activated protein

kinases (MAPK) and the transcription factors NFκB and AP-1. NE (10 µM) induced a

transient increase in ERK and SAPK/JNK phosphorylation, as assessed by western

blotting (Figure 6A). NE activated NFκB, as indicated by increas NFκB-dependent

gene expression as well as nuclear translocation of the heterodimer p50/p65 (Figure 6B

and 6C). In addition, NE increased tumor necrosis factor α (TNF-α)-induced NFκB

activation, suggesting that it could amplify the inflammatory response evoked by other

cytokines (data not shown). Finally, NE stimulated AP-1 DNA binding, as assessed by

EMSA (Figure 6D). To elucidate the role of these signaling pathways on chemokine

production, HSC were preincubated with specific antagonists before NE stimulation.

Preincubation of cells with BAY11-7082 (a NFκB inhibitor), but not with SP600125 (a

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JNK inhibitor) and PD231445 (an ERK inhibitor), attenuated IL-8 secretion (Figure.

5B). Moreover, transfection with Ad5IκB reduced IL-8 production confirming NF-κB

involvement (Figure. 5C). These data strongly indicate that NE exerts pro-inflammatory

actions in human HSC through NFκB activation.

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DISCUSSION

In the current study, we investigate the biological effects of NE in human HSCs.

We provide evidence that HSC express some α and β receptor subtypes.

Norepinephrine stimulates key intracellular signaling pathways in these cells and

induces cell contraction and proinflammatory actions. These actions are mainly

mediated by α1 receptors, although β receptors also modulate some of the effects

evoked by NE. Moreover, α1 receptors are markedly up-regulated in human fibrotic

livers. These results suggest that HSC are potential targets for the pathogenic effect of

catecholamines in the liver and in keeping with recent data indicating that

catecholamines not only regulate vascular homeostasis, but also contribute to tissue

repair and inflammation (11; 15; 30; 31).

The hallmark biological action of NE in vascular cell types is to induce Ca2+

increase and cell contraction. This effect has been extensively studied in mesangial cells

and vascular smooth muscle cells (18; 26). Similarly, a large number of studies have

investigated the vasoactive effects of vasoconstrictors in cultured human HSC,

including angiotensin II, endothelin-1, thrombin and leukotriene D4 (36). Currently,

studies using activated HSC are widely used to investigate the cellular basis of portal

hypertension in order to identify potential targets for therapy. Surprisingly, little

information is available on the actions of adrenergic agonists in these cells. Here, we

provide evidence that NE is a potent agonist for human HSC to induce [Ca2+]i increase.

An intriguing finding of the current study is that the Ca2+ response pattern evoked by

NE markedly differ to that observed with other vasoconstrictors. Previously, we have

shown that angiotensin II induces an abrupt [Ca2+]i increase followed by a sustained

phase (6). This effect was mainly due to the entrance of Ca2+ from the extracellular

space through voltage-operated calcium channels. Interestingly, the effect of NE on

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[Ca2+]i consists of numerous Ca2+ spikes. Baseline Ca2+ spikes are characterized by

rapidly-rising transient increases in [Ca2+]i rising from a baseline which is close to the

resting concentration (9). This pattern of response has been described in many cells in

response to hormones and neurotransmitters (18; 25). Ca2+ oscillations permit cells to

respond to agonists without being exposed to sustained levels of [Ca2+]i. It has been

shown that oscillations and specially their frequency activate cell signaling like

calmodulin-dependent protein kinase II and NFκB (14; 22). Moreover, Ca2+ oscillations

have been involved in many cellular processes like contraction, migration, cell secretion

or phagocytosis (9; 12). The biological relevance of NE-induced Ca2+ spikes in HSC is

unknown. Although NE induced MLC-II phosphorylation, a prerequisite for cell

contraction, and induced cell contraction, it failed to induce cell proliferation, which is

commonly stimulated by vasoconstrictors in HSC. It is possible that sustained [Ca2+]i

increase, rather that [Ca2+]i oscillations, are required to induce cell proliferation. Further

studies should investigate the pathophysiological relevance of agonist-induced Ca2+

spikes in HSC.

A relevant finding of the current study is that NE stimulates key intracellular

signaling pathways implicated in the pathogenesis of hepatic inflammation and

fibrogenesis. These pathways include NFκB, JNK/AP-1 and ERK. Importantly, NE

enhances chemokine secretion in a NFκB-dependent manner. Besides their role in

collagen synthesis, recent data suggest that activated HSC play a supportive role in the

inflammatory response to injury (27). These cells proliferate at the areas of liver injury

and a cross-talk between HSC and infiltrating leukocytes is likely to occur. HSC can

present antigens, express cell adhesion molecules, regulate lymphocyte proliferation and

secrete powerful chemokines such as IL-8 and RANTES (27; 42). On the other hand,

mediators derived from lymphocytes (e.g. CD40 ligand, RANTES) can regulate HSC

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biological functions and stimulate their fibrogenic potential (40; 41). Therefore, by

stimulating chemokine secretion, NE may enhance inflammation in chronic liver

diseases. Whether NE also exerts proinflammatory actions in other non-parenchymal

hepatic cells types (i.e. Kupffer cells and sinusoidal endothelial cells) is unknown and

deserves further investigation.

In the current study, despite the possible proinflammatory effect of NE, we did

not find a direct fibrogenic effect of NE on collagen expression. In rodent early-cultured

HSC, NE stimulates collagen secretion, suggesting that it directly induces fibrogenic

effects in these cells (32; 33). These discrepant results can be explained by the fact that

we used passaged fully-activated human HSC. In this cell phenotype, collagen is highly

expressed and fibrogenic agonists commonly fail to further increase its expression.

Other biological functions, such as chemokine secretion, can be stimulated by

inflammatory mediators (40; 41). However, it is well-known that liver inflammation

precedes and promotes the progression of liver fibrosis and there is a positive

correlation between the degree of sustained hepatic inflammation and the progression of

fibrosis. Moreover, substances inhibiting the inflammatory response of the liver also

limit fibrogenesis (5). Therefore, we speculate that the proinflammatory effect of NE on

HSC may play a role in promoting hepatic inflammation in patients with chronic liver

diseases, thus favoring fibrosis development.

The biological effects of NE in HSC may have pathophysiological

consequences. First, contraction of activated HSC are believed to increase intrahepatic

vascular resistance in the cirrhotic liver and contribute to the pathogenesis of portal

hypertension (37). The finding that NE induces contraction in HSC may explain, at least

in part, the effects of catecholamines in regulating intrahepatic vascular resistance in

cirrhosis. Moreover, the fact that HSC synthesize NE suggest that not only an endocrine

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pathways may regulate HSC contractility but also an autocrine/paracrine system may be

involved (32). Further studies should study this hypothesis. Our finding that NE

enhances chemokine secretion in HSC suggests that catecholamines may exert

proinflammatory effects in the liver. The pro inflammatory effect of NE on HSC has not

been previously described in other cell types. The finding that NE may promotes

proinflammatory actions in HSC expands the traditional view of the SNS as a system

that basically regulates systemic and hepatic hemodynamics.

In summary, the results of the current study indicate that NE mediates vasoactive

and proinflammatory actions in human HSC mainly through α1 adrenoceptors. Because

HSC are able to synthesize adrenergic agonists and the SNS is activated in patients with

advanced liver fibrosis, these findings suggest that catecholamines may increase

intrahepatic vascular resistance and promote inflammatiory actions by targeting HSC.

Our data explain, at least in part, the mechanisms underlying the beneficial effects of α1

receptor antagonists in the treatment of portal hypertension and suggest the potential use

of these drugs to modulate hepatic inflammation and subsequent fibrogenesis.

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Table 1. PCR primer pairs for adrenoceptors and housekeeping genes. Gene Sense Antisense Product (bp)GADPH 5’-CCATGGAGAAGGCTGGGG-3’ 5’-CAAAGTTGTCATGGATGACC-3’ 198 α1A- 5’-TGGCCGACCTCCTGCTCACCTC-3’ 5’-GGCCCCGGCTTTCCCTCTTG-3’ 444 α1B- 5’-CCCCCGACGCCGTGTTCAAGGTG-3’ 5’-CTCAGGCGCGGGCAGGCTCAGGA-3’ 407 α2A- 5’-GCCGGCCTGCTCATGCTGCTCAC-3’ 5’-GCCGCCGCCGCCCTTCTTCTC-3’ 420 α2B- 5’-CAGGCCGATGGAGGTGGTTCAAAT-3’ 5’-CCGGAGGGGTGCTGGGTAAGGA-3’. 423 β1- 5’-GTGGCCCTACGCGAGCAGAA-3’ 5’-GCGGCAGTAGATGATGGGGTTGAA-3’ 195 β2- 5’-TCTGGCCGCGTTTCTGTGTTGG-3’ 5’-GGCCCCGGCTTTCCCTCTTG-3’ 295

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Table 2. Effect of norepinephrine (NE) on [Ca2+]i in human hepatic stellate cells n Responder

cells (%)a Peak [Ca2+]i

(nM)b Oscillations (% of cells)

NE 10 µM 51 84 317±138 57

Prazosin 1 µM + NE 10 µM 27 0 - - Propranolol 1 µM + NE 10 µM 69 79 260±170* 36* aResponder cells are those showing at least a 1.5 fold increase in [Ca2+]i compared to resting values. bResponders cells only. Values are given as mean ± SEM. *p<0.05 vs cells stimulated with NE.

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FIGURE LEGENDS

Figure 1. Expression of adrenoceptors in human hepatic stellate cells (HSC). α1A,

α2B, and β2 receptors were detected in HSC and whole liver, as assessed by RT-PCR.

α1B, α2A and β1 receptor were not detected in HSC but were amplified in mRNA

isolated from human livers (A). GAPDH was amplified as a housekeeping gene to

assess correct amplification (data not shown). Detection of α1A receptor in HSC freshly

isolated from normal human livers (quiescent) (B). Green staining shows α1A receptor

expression. Detection of α1A receptor in HSC activated after prolonged culture (C) by

immunocytochemistry. Note α1A adrenoceptor expression at cell membrane (arrow). A

negative control using a isotype-matched primary antibody was performed (C).

Figure 2. Expression of α1A adrenoceptors in normal and cirrhotic human livers.

Western blot analysis of 50 µg proteins from normal and fibrotic human livers. α1A

adrenoceptors were detected in normal livers and were overexpressed in cirrhotic livers

(51.5 KDa). Actin expression was performed by Western blotting to ensure equal

protein loading (A). Analysis of α1A receptor expression by densitometry respect to

actin (B). **p<0.01 vs normal livers.

Figure 3. Effects of norepinephrine (NE) on [Ca2+]i and cell contraction in hepatic

stellate cells (HSC). Representative graph showing the effect of NE in a single HSC.

Norepinephrine (10µM) induced an oscillatory [Ca2+]i increase (Ca2+ spikes) (A).

Preincubation with prazosin (1 µM) for 10 min blocked NE-induced [Ca2+]i increase

(B). Incubation for 10 min with propranolol (1 µM) decreased both Ca2+ peak and

percentage of cells with oscillations (C). Representative images of [Ca2+]i increase

induced by NE (10µM) in HSC (D). Images were obtained using a confocal microscope

in fluo-4 loaded HSC. Images were taken every 10 seconds. Color change into red

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reveals [Ca2+]i increase. Phosphorylation of myosin light chain-II (MLC-II), as assessed

by western blotting (18 KDa). Stimulation with NE (10 µM) or Ang II induced MLC-II

phosphorylation at 5 min (E). Representative images of wrinkle formation (arrows)

formed by HSC stimulated with NE (10 µM) (F).

Figure 4. Effect of norepinephrine (NE) on cell growth and collagen gene

expression. Effect of NE (10µM) on cell growth, as assessed by 3H-thymidine

incorporation (A). Hepatic stellate cells were treated for 24h with NE (100µM-10nM),

platelet derived growth factor-BB (PDGF-BB, 20 ng/ml) or both. PDGF-BB induced a

marked increased in DNA synthesis (*p<.001 vs untreated cells). Norepinephrine did

not stimulate cell growth nor amplified PDGF-induced mitogenic effect. Effect of NE

(10 µM) on procollagen α1(I) gene expression, as assessed by quantitative PCR (B).

Figures are the mean of 5 independent experiments.

Figure 5. Effect of norepineprhine (NE) on chemokine secretion in human hepatic

stellate cells (HSC). (A) Norepinephrine stimulated the secretion of interleukin-8 (IL-8)

(■) and RANTES (□) to the culture media, as assessed by specific ELISA. Incubation

with prazosin prevented NE-induced chemokine secretion. Propranolol reduced NE-

induced RANTES secretion. (B) Preincubation with BAY11-7082 (a NFκB inhibitor),

but not with SP600125 (a JNK inhibitor) and PD231445 (a ERK inhibitor) prevented

NE induced IL-8 secretion. (C) Ad5IκB reduced IL-8 production induced by NE.

*p<0.05 vs untreated cells Ad5GFP was used as a control. #p<0.05 vs cells treated with

NE alone (n=5).

Figure 6. Effect of norepineprhine (NE) on intracellular signaling pathways in

human hepatic stellate cells (HSC). (A) Effect of NE on ERK and SAPK/JNK

phosphorylation, as assessed by western blotting. Stimulation with NE induced a

transient phosphorylation of ERK and SAPK/JNK. Figures are representative of three

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independent experiments. (B) NFκB dependent gene expression, as assessed by a

luciferase assay. Norepinephrine stimulated NFκB-dependent gene expression that was

completely blunted by prazosin, but not by propranolol. *p<0.05 vs untreated cells.

#p<0.05 vs cells treated with NE alone. Results are the mean of 4 independent

experiments. (C) Immunocytochemistry analysis of the p65 subunit of NFκB in HSC.

Stimulation for 60 min with NE (10µM) resulted in the nuclear translocation of the

heterodimer p50/p65 to the nucleus, which is indicative of NFκB activation. TNFα (10

ng/ml) was used as a positive control. (D) Activation of the transcription factor AP-1, as

assessed by EMSA. Stimulation with NE (10 µM) for 60 min markedly stimulated AP-1

DNA binding. Preincubation with cold probe was performed to ensure specificity. This

figure is representative of 3 independent experiments.

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Abbreviations.

AP-1, activating protein-1; EMSA, electrophoretic mobility shift assay; ERK,

extracellular regulated kinase; HSC, hepatic stellate cells; IL-8, interleukin-8; [Ca2+]i,

intracellular calcium concentration; JNK, Jun-N-terminal kinase; MAPK, mitogen-

activated protein kinase; MLC-II, myosin light chain-II; NFκB, nuclear factor kappa B;

NE, norepinephrine; PDGF, platelet derived growth factor-BB;SNS, sympathetic

nervous system; TNF-α, tumor necrosis factor α.

Acknowledgements.

We thank Joan Carles Garcia-Pagán for helpful discussion, Elena Juez and Cristina

Millán for her excellent technical support. We thank Maria Calvo and Anna Bosch

(Serveis Cientificotècnics, Universitat de Barcelona- IDIBAPS) for excellent technical

assistance in in vivo confocal microscopy. This work is supported by grants from the

NIH (R01 AA15055), from the Ministerio de Ciencia y Tecnología, Dirección General

de Investigación (SAF2002-03696), and from the Instituto de Salud Carlos III

(CO3/02). Pau Sancho-Bru had a grant from the Institut d’Investigacions Biomèdiques

August Pi i Sunyer (IDIBAPS). Montserrat Moreno had a grant from the Instituto Reina

Sofía de Investigación Nefrológica.

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Short Title: Effects of Norepinephrine In Hepatic Stellate Cells

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