mollecular hepatic stellate cells targets in

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MOLLECULAR HEPATIC STELLATE CELLS TARGETS IN

ANTIFIBROTIC THERAPY

1 1*Dr. Alin Gabriel Ionescu , Conf. Univ. Dr. Cristin Constantin Vere ,

1 1 1Dr. Costin Teodor Streba , Dr. Liliana Streba , Prof. Univ. Dr. Ion Rogoveanu

1University of Medicine and Pharmacy, Craiova

ABSTRACT. Hepatic stellate cells (HSC) activation as a result of liver injury, followed by their proliferation andthe increase of extracellular matrix (ECM) synthesis, is the main phenomenon in the development of liverfibrosis. In case of chronic liver injury, the negative consequences generated by liver fibrosis on hepatic functionand morphology may lead to hepatic insufficiency and portal hypertension. Liver cirrhosis is considered aprecancerous stage prior to the development of hepatocellular carcinoma (HCC). Initiation of HSCs activationconsists in rapid changes in protein synthesis, followed by alterations in cellular phenotype, amplifying HSCsresponse to inflammatory cytokines and local stimuli. By blocking activated HSCs response to inflammatory

cytokines and growth factors stimulation, antifibrotic therapies aim to inhibit their proliferation, trigger theirapoptosis, reduce the collagen synthesis and increase ECM degradation. Future therapeutic strategies targetthe development of oral drugs with high antifibrotic potential, as well as a series of therapeutic agents withparenteral administration and reduced side effects. Also, a special attention should be given to gene therapy thatalters HSCs phenotype and inhibits exacerbated collagen synthesis. In the same time, prophylactic andneoadjuvant therapy for HCC aims to block activated HSCs expression of growth and nuclear factors involved incarcinogenesis.

Keywords:hepatic stellate cells, liver fibrosis, antifibrotic therapy, hepatocellular carcinoma, carcinogenesisnotknown.

INTRODUCTION Initiating fibrogenesis

Hepatic injury initiates fibrogenesis throughLiver fibrosis and cirrhosis develop after chronic liversignalling molecules derived from inflammatory cells,injuries of different ethiologies viral, autoimmune,hepatocytes and other non-parenchymal cells, especiallydrugs, cholestasis and metabolic. Excessivesinusoidal endothelial cells and Kupffer cells.

accumulation of ECM is present in most chronic liverOxidative stress plays an important role in hepatic

diseases. In injured hepatic tissue, activated HSCs are theinjury and in initiating liver fibrogenesis through

main collagen producing cells. Following liver injury, production of reactive oxygen species (ROS) .quiescent HSCs activates and suffer a phenotypic Hepatocytes' necrosis and apoptosis appear followingtransdifferentiation in proliferative, fibrogenic and lipids, proteins and DNA oxidation, followed by

contractile myofibroblasts-like cells. amplifying inflammatory response and initiating

fibrogenesis. ROS stimulates both Kupffer andBeside hepatoprotection and treatment of the primaryinflammatory cells in releasing profibrogenic mediators,disease, most antifibrotic therapies target the inhibition

that in turn stimulates HSCs proliferation whichof HSCs activation by reducing inflammation or host'samplifies the production of ECM (Galli et al., 2005).

immune inflammatory response, as well as neutralisingHypoxia leads to fibrosis by stimulating the release of

HSCs proliferative, profibrogenic, contractile and/orHypoxia-inducible factor 1 (HIF)-1 by HSCs. In turn,

proinflammatory response. A different therapy takes into (HIF)-1 stimulates the expression of vascularaccount triggering HSCs apoptosis. endothelial cell growth factor (VEGF), which leads to an

Other antifibrotic therapies consider direct increase of type I collagen synthesis by HSCs (Corpechot

administration of collagenases like matrix- et al., 2002). Hypoxia intervenes in autocrine and

paracrine control of angiogenesis and fibrosis bymetalloproteases (MMPs), or stimulation of MMPsincreasing the stimulation and synthesis of transformingproducing cells, as well as blocking tisular inhibitors ofgrowth factor (TGF)-1 (Jeong et al., 2004). Followingmetalloproteases (TIMPs) which exert an inhibitorychronic liver damage, a paracrine loop between fibrosis

effect on MMPs, leading to an increased ECM

and hypoxia is created, as they reciprocally stimulatedegradation. each other and therefore disrupt normal tissue repair.

*Correspondence: Conf. Univ. Dr. Cristin Constantin Vere, University of Medicine and Pharmacy Craiova, Faculty of Medicine,Department of Internal Medicine, no. 2-4 Petru Rares St., 200349, Dolj, Romania, Tel. +40-(251)- 522 458,Fax. +40-(251)- 593 077, email: [email protected]

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Chronic hepatic injury generated by hepatitic viruses,

Inflammation and immune response play an toxins, autoimmune diseases stimulates the activation of

important role in the initiation and progression of liver HSCs through cytokines, which leads to their

fibrosis (Mehal et al., 2007). Chronic liver inflammation transformation into myofibroblast-like cells (Bataller et

leads to the activation of Kupffer cells which locally al., 2005). HSCs activation comprises two major phases:

release proinflammatory cytokines like tumoral necrosis initiation (preimflammatory stage) and perpetuation,

factor (TNF)-, interleukin-1 (IL-1) and interleukin-6 followed by resolution, if case hepatic injury ends

(IL-6) (Capuron et al., 2011; Laye et al., 2000). (Friedman, 2004).

Electron microscopic examination revealed that the Initiation represents the early genetic and structural

vacuolar juice with anthocyans undergoes a fine alteration of HSCs, followed by emphasizing their

granulation of their vacuolar content; witch can be a response to cytokine stimulation, shortly after hepatic

result of samples preparation with osmic acid. It is not injury occurs. Initial paracrine stimulation which

clear if leucoantocians accumulated in vacuolele cells of includes signals from injured hepatocytes, Kupffer and

root tissue undergo this fine granulation, or only those endothelial cells, leads to early HSCs activation, as well

with red anthocyanins. Future researches are needed to as alteration in surrounding ECM structure. Once the

elucidate this phenomenon. activation process is initiated, perpetuation follows after

Kupffer cells activation emphasizes the activity of continuous stimulation which in turn maintains HSCs in

nuclear factor kappa-B (NF-kB) with a subsequent active state, involving seven alterations in cellular

release of proinflammatory cytokines, including TNF- behaviour: proliferation, chemotaxis, fibrogenesis,

and the monocyte chemoattractant protein (MCP)-1, contractility, ECM degradation, retinoid loss and

which initiates HSCs activation (Liu et al., 2010). leucocite chemoattraction / cytokine release. During

In turn, HSCs respond to this stimulation by releasing perpetuation, a paracrine and autocrine release of

macrophage colony-stimulating factor (M-CSF), MCP-1 proinflammatory, profibrogenic and promitogenic

(Czaja et al., 1994) and IL-6, (Tiggelman et al., 1995) stimuli occurs.

which amplifies acute phase response and leads to a

supplementary increase of activated macrophages. TNF- Initiation

also induces neutrophil infiltration locally in hepatic Initial alterations in HSCs structure appear following

injury and increases mitochondrial oxidation in paracrine stimulation generated by Kupffer cells,

hepatocytes, initiating their apoptosis (Pinzani et al., lymphocytes, hepatocytes, leucocytes and sinusoidal

1992). Hepa tocyte s apop to si s in te rvenes in endothelium. A major role in HSCs activation is played

proinflammatory stimulation and in fibrogenesis through by Kupffer cells infiltration and activation (Gressner etapoptotic bodies which are phagocited by Kupffer cells al., 1993), which in turn stimulates cellular proliferation,

that subsequently synthesizes TNF- (Canbay et al., ECM synthesis and retinoid loss, via TGF- (Matsuoka et

2003). Similarly, HSCs induces profibrogenic response al., 1990), TNF- and matrix metallo-proteases (MMP)-9

after assimilating apoptotic bodies, through producing (Winwood et al., 1995). Endothelial cells participate in

oxidative radicals, up-regulating TGF-1 synthesis and HSCs activation by converting TGF- from passive to

type I collagen expression (Zhan et al., 2006). profibrogenic active state, and also by increasing

Hepatic steatosis indirectly generates liver fibrosis, synthesis of cellular fibronectine.

by increasing hepatocyte Kupffer cells generate locally, in the injured hepatic

susceptibility to oxidative stress and viral infection tissue, on the one hand ROS which stimulated HSCs

aggression, both leading to HSCs activation followed by activation and collagen I synthesis, and on the other hand

an increased accumulation of ECM. A series of different they produce nitric oxide (NO) which exerts an

pa thwa ys may cont ribu te to st ea to si s- re la te d antagonist effect of ROS, diminishing HSCsfibrogenesis, as increasing oxidative stress response, proliferation and contractility. A study conducted on a co-

increasing hepatocytes susceptibility to apoptosis, culture of CYP2E1 cells virally infected with HepG2 and

alteration in cellular response to injury, signalling and HSCs, has shown that hepatocytes-generated ROS can

activation of peroxisome proliferator-activated receptor increase the production of collagen by activated HSCs

(PPAR), as well as dysregulation of leptin expression and via P4502E1 cytochrome (Nieto et al., 2002). A series of

signalling (Friedman, 2003). clinical studies have reported a diminishing in activated

HSCs number, following a treatment with antioxidant

HSCs activation and myofibroblast transformation inhibitors of CYP2E1 and hydrogen peroxidase. NADPH

A major breakthrough in understanding the oxidase represents another ROS source generated by

mechanism of liver fibrosis was the identification of injured hepatocytes which in turn stimulate fagocites to

HSCs role. HSCs are the main cells involved in produce superoxide anions. Hepatocyte apoptosis also

producing and deposition of ECM in normal and fibrotic stimulates HSCs activation, followed by liver fibrosisliver. development (Canbay et al., 2003).

(Radu et al., 2011),

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An important source of ROS is represented by monocytes MCP-1 (Marra et al., 1998). HSCs synthesizeneutrophiles stimulating collagen I synthesis by activated multiple anti-inflammatory cytokines, especially IL-10.HSCs via superoxidation. Neutrophiles produce in the During perpetuation, ECM remodelling continues,same time NO, which reduces superoxide effect, without low density subendothelial matrix being progressivelycancelling it (Casini et al., 1997). replaced by an ECM rich in fibril-producing collagen,

T-hepler (Th) CD4 lymphocytes intervene in HSCs which leads to an altered behaviour of hepatocytes,activation by differentiating in subpopulations Th1 and sinusoidal endothelium cells and HSCs. In advancedTh2 and also by cytokine synthesis. Th1 cells release - fibrosis stages, in hepatic tissue there has been identifiedinterferon (INF), TNF and IL-2, and also produce an ECM concentration 6 times greater than normal,cytokines with roles in cellular-mediated immunity. In including collagen I, III and IV, undulin, laminin,turn, Th2 cells synthesize IL-4, IL-5, IL-6 and IL-13, proteoglycans, fibronectin, elastin and hyaluronan.promoting humoral-mediated immunity which increases Fibril-producing ECM accelerates HSCs activationliver fibrosis. During viral aggression, leucocytes through integrins and by binding receptors located in thepresents in the liver join Kupffer cells and synthesize discoidin domain receptor (DDR2). DDR2 is a tyrosinecytokines which modulate HSCs phenotype. kinase receptor, ligand for type I collagen. After binding,

HSCs trigger innate immune response through a sequence of events is triggered, including src kinaseinflammatory signalling of Toll-like receptors (TLR4) attraction, followed by signal amplification andwhich are part of pattern recognition receptors family. transcriptional induction of MMP-2 (Ikeda et al., 2002).

TLR4 receptors have been identified on HSCs andKupffer cells surfaces. An animal model study has shown HSCs perpetuationa reduction in local macrophage infiltration and an The perpetuation of HSCs activation involved a seriesimprovement in liver fibrosis, which had been of alterations in cellular behaviour, like proliferation,experimentally induced by genetic deletion of TLR4 cont rac ti li ty, fibr ogenes is , ECM deg radat ion,receptor. As a result of TLR4 receptor blockage by chemotaxis and retinoid loss.liposaccharide ligands (Paik et al., 2003), an intracellularsignalling pathway is activated, which includes NF-kB Proliferationactivation (Szabo et al., 2006). Early alterations in ECM Local activated HSCs proliferation follows growthstructure are consequences of transcriptional events. factor stimulation. PDGF exerts the most potent mitogen

GC, GT sequences and CACCC boxes are present in effect towards HSCs. In early stages of HSCs activation,regulatory regions of profibrogenic genes expressed in PDGF receptors are expressed on cell surface,activated HSCs, where are located genes receptors for 1 amplifying the responsiveness to its stimulation (Wong et(I) collagen, TGF-1 and TGF- type I and type II.

al., 1994). Its experimental inhibition has been followedThese sequences encode receptors for transcription by a reduction in the degree of liver fibrosis. In hepaticfactor Krupple-like (KLF), which contains terminal DNA injured tissue, HSCs proliferate after PDGF stimulation,C2H2C. This transcription factors family modulates accomplished through mitogen activated protein kinasespecific tissular genes transcription. In activated HSCs, (MAPK) signalling, especially JNK, extracellular signal-there have been identified at least three members of this regulated kinase (ERK) and p38. JNK and ERKtranscriptional type factors Sp1, basic transcription activation induces HSCs proliferation, while p38element binding protein (BTEB)-1 and KLF6 which can activation inhibits proliferative response (Schnabl et al.,modulate collagen 1 (I) gene transcription. In vivo and in 2001).vitro studies have reported an early presence of KLF-6 PDGF stimulates the activation of AKt kinase and p70gene subsequent to HSCs activation (Bieker, 2001). In in activated HSCs, by means of phosphatidylinositol 3activated HSCs, SP1 plays the role of a transcriptional kinase (PI3-K), which leads to an exacerbation of HSCsmodulator (Rippe et al., 1995) associated to the two proliferation and chemotaxis (Reif et al., 2003). ET-1,regions GC (FP1 and FP2) belonging to collagen 1 (I) thrombin, FGF, insulin-like growth factor (IGF) also

promoter gene (Rippe et al., 1999). exerts a mitogen effect on HSCs (Pinzani et al., 2001).

Perpetuation ContractilityParacrine stimulation is present during HSCs The increase in portal resistance in injured liver is

perpetuation, process which involves simultaneous generated by activated HSCs contractility. ET-1 and otherautocrine cytokines which triggers HSCs activation, like contraction-stimulating cytokines favour HSCsTGF-b1, platelet derived growth factor (PDGF), contraction. An increased production of ET-1 isfibroblastic growth factor (FGF) and endothelin-1 (ET- accompanied by increased levels of the conversion1), as well as inhibitory cytokines of HSCs activation like enzyme of ET-1 which activates inactive ET-1.hepatocyte growth factor (HGF). Endothelin stimulates HSCs through two G protein

HSCs amplify the inflammation of hepatic lesion by coupled receptors of endothelin type A and B (ETA andreleasing chemoattractants (Marra et al., 1993) for ETB) present on quiescent and active HSCs surfaceneutrophi les cytokine- induced neutrophi l (Rockey, 1997). In intralobullar spaces, ET-1 binding

chemoattractant / IL-8 (Maher et al., 1998) and for points are abundant especially in the juxtaportal region.

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An ultrast ructural analysis has shown that HSCs inhibits the activity of interstitial collagenases,

aproximatively 35% of ET-1 binding points are located leading to scar tissue accumulation (Arthur, 1995).

on the HSCs surface, and only a part of the other binding During fibrogenesis, MMP-2, type I membranar

points are located on endothelial sinusoidal cells (ESC) MMP, TIMP-1 and TIMP-2 concentrations are increased

and Kupffer cells (Gondo et al., 1993). (Arthur, 2000). On activated HSCs and myofibroblasts

The heterogeneity of the ET-1 binding places in there is an increased concentration of TIMPs. Liver

hepatic lobules follows blood vessels tracts in hepatic macrophages are a source of MMP-13 (Uchinami et al.,

sinusoids. All cell types present in hepatic sinusoids 2006) and proteases (Hironaka et al., 2000). Clinical

Kupffer cells, ESCs, HSCs and hepatocytes express ETB studies have emphasized that increased levels of MMPs

receptors, but only HSCs and hepatocytes express ETA reduce liver fibrosis, but not biliary fibrosis (Garcia-

receptors (Mallat et al., 1995). ET-1 induces HSCs Banuelos et al., 2002), thus demonstrating MMPs

contraction via ETA receptors (Shi-Wen et al., 2004). NO different role depending on type and duration of injury.

is an ET-1 antagonist, stimulating HSCs relaxation. Activating protein (AP)-1 modulated genetic

remodelling of TIMP and ECM, presenting an increased

and persistent activity in activated HSCs). AP-1 containsFibrogenesisat least one Jun type protein which may form homodimersStimulation of collagen synthesis is mediated byor heterodimers with other Jun proteins or with a Fos typetranscriptional and posttranscriptional mechanisms, and

protein. JunD protein is an important protein of AP-1,it represents one of the major molecular responses of with a major role in genetic stimulation of TIMP-1 andactivated HSCs following hepatic injury. Collagen IIL-6 synthesis (Smart et al., 2001) in activated HSCs,transcriptional activation is mediated by fibrogenicTIMP-1 transcription being regulated by JunDgrowth factors (Gressner, 1995), adipokines andhomodimers operating in parallel with a nuclear RUNXvasoactive substances, needed in fibrosis development.protein (Trim et al., 2000).Following TGF-1 stimulation, activated HSCs increase

As a response to cellular microenvironmentECM synthesis (Olaso et al., 1998). HSCs represent asignalling, alteration in concentration and activity ofmajor source of TGF-1, which is also synthesized bydifferent types of AP-1 and in the genes they transcriptKupffer cells and platelets.(MMPs and TIMPs), stimulates liver ECM remodellingIncreased levels of TGF-1 as a result of liver fibrosisby activated HSCs.stimulates HSCs proliferation through Smad and MAPK

signalling pathways (Cao et al., 2002), as well as collagen

1 (I) synthesis by activated HSCs (Dooley et al., 2001). HSCs chemotaxis

In activated HSCs, TGF- increases 1 (I) collagen Following activated HSCs migration, their numbermRNA half-time mediated by increasing stability of 1 (I) increases in the areas of injury. Many chemoattractants

collagen mRNA through p38 MAPK pathway (Tsukada are involved in HSCs migration, like PDGF, IGF-1,

et al., 2005). The stability of 1 (I) collagen is increased MCP-1 and ET-1. MCP-1. MCP-1 is a chemokine

due to the interaction between a stem loop structure synthesised only by activated HSCs, and it signals

located at the 5' end of 1 (I) collagen with an untranslated through PI3-K mediated pathways with Ca2+ influx the

3' region (Lindquist et al., 2004). accumulation of lymphocytes and monocytes in the area

Experimentally, TGF- involvement has been reported of injury.

mainly in perpetuation and less in initiating HSCs

activation. Animal model studies have shown that Retinoid lossblocking TGF- synthesis or its signalling pathways An important feature of HSCs activation isprevents fibrosis development (Shek et al., 2004). represented by the loss of its intracellular retinoid.

Leptine is an adipokine mainly synthesised by Although one cannot firmly state that this loss isadipocytes. Its role as a profibrinogenic hormone was necessary or not for HSCs activation, Milliano et al. haveemphasized based on an increased synthesis of 1 (I) reported that HSCs exposed to extracellular retinoidcollagen by activated HSCs, mediated through PI3K/AKt stimulation present a decrease of activity markers,pathway stimulated by activated JAK1 kinases (Niu et without stopping their activation (Milliano et al., 2005).al., 2007). Multiple nuclear receptors for retinoids were

identified on the surface of activated HSCs (Fiorucci et

ECM degradation al., 2005).

Fibrosis resolution is characterized by increased PPAR receptors , especially PPAR, have been

collagenolitic activity, through degradation of fibrillar identified on HSCs membrane (Marra et al., 2000), their

collagen I and III by interstitial MMPs. HSCs contain all number increasing proportionally with cellular activity

components needed for ECM degradation, being a MMP- (Hellemans et al., 2003). Marra et al. have shown that

2 source as well as MMP-3 / stromelysin. During liver ligands blocking the PPAR nuclear receptor reduce HSCs

fibrosis, due to a reduced level of TIMP-1, the activity of activation, while ligands of PPAR receptor stimulateMMPs increases. By TIMP-1 and TIMP-2 stimulation, HSCs proliferation (Marra et al., 2000).

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Resolution HSCs involvement in hepatocellular carcinoma

During tisular healing of acute hepatic injury, it has developmentbeen observed a reduced number of activated HSCs, as a HSCs activation plays an important role in theresult of reversion into quiescent state, or apoptosis progression of liver fibrosis, being the main risk factor ininitiation. Due to this observation, antifibrotic therapies HCC development (Elsharkawy et al., 2007).

of chronic hepatic injuries regard stimulating the HSCs transdifferentiation in myofibroblasts-likepathways which lead to HSCs reversion in quiescent state cells is responsible for an excessive ECM production andor inducing their apoptosis. for replacing normal liver tissue. An increased number of

activates HSCs has been observed around tumorReversion sinusoids, fibrous septa (Schmitt-Griff et al., 1991), as

PPAR is a member of nuclear receptors of steroid or well as in the tumor capsule (Enzan et al., 1994).thyroid hormones superfamily which plays a role in HSCs activation plays a major role ingenetic transcription control and cellular differentiation. hepatocarcinogenesis (Zhao et al., 2011) by inducingIn vivo and in vitro studies have reported a reduction of autocrine signalling of TGF- and by stimulating -cateninPPAR synthesis in activated HSCs (Miyahara et al., nuclear accumulation in neoplastic hepatocytes (Mikula2000). Hazra et al. have created cultured activated HSCs et al., 2006). TGF- releasing by the activated HSCsand they have demonstrated their reversion throughout an stimulates tumor progression in neoplastic hepatocytes.increased synthesis of PPAR. PPAR inhibits the TGF- induces epithelial cells transdifferentiation in

activation of a1 (I) procollagen promoter and it reduces mesenchymal cells and it amplifies PDGF signalling inthe collagen synthesis, thus determining the reversion of oncogenic Ras transformed hepatocytes. One hypothesisbiochemical characteristics of activated HSCs (Hazra et regarding HCC tumorogenesis implies the combinedal., 2004). effect of growth factor FGF-1 and -2, PDGF and IGF

released by activated HSCs (Bataller et al., 2005).In HCC, activated HSCs considerably increase NF-Apoptosis

kB and ERK activity. NF-kB and MAP kinase / ERKIn vitro studies have reported the induction ofpathways are involved in HCC progression byactivated HSCs apoptosis following membranarstimulating tumor cells proliferation and by inhibitingreceptors stimulation by apoptosis-inducing membranarapoptosis (Amann et al., 2009). HSCs intervene inproteins TRAIL and CD95-L (Radaeva et al., 2006).tumoral metastasis via HGF which amplifies tumoralNervous growth factor also stimulates HSCs apoptosis,cells invasiveness. A study which used antagonists ofits effect being antagonised by serotonin (Ruddell et al.,tumoral cells HGF receptors, reported that tumoral cells2006).development and migration were impaired (Barnaeva etNatural killer (NK) cells intervene in fibrosisal., 2007).resolution and HSCs apoptosis through the release of

Activated HSCs also intervene in tumoralTRAIL proteins. NK cells' antifibrotic role wasprogression, by alterations in ECM remodelling (Thretconfirmed by clinical trials which demonstrated anet al., 2001) and by releasing angiopoietin which inducesintensification of liver fibrosis after the interruption ofangiogenesis (Wirz et al., 2008). VEGF released byimmunosuppressive therapies (Hudnall, 1991). Duringtumoral cells and activated HSCs is involved inliver fibrosis regression, senescent HSCs are moreangiogenesis associated with HCC progressionsusceptible to NK cells induced apoptosis (Krizhanovsky(Torimura et al., 1998).et al., 2008).

These evidences promote the pathways throughDuring the healing phase of experimentally inducedwhich HSCs take part in HCC development andliver injury, the association between HSCs apoptosis andprogression as new therapeutic strategies in HCCreduced levels of TIMP-1 synthesis has beentreatment.demonstrated (Iredale et al., 1998). TIMP-1 generates an

anti-apoptotic effect, thus protecting activated HSCs.

Blocking TIMP-1 with specific monoclonal antibodies is Curing the primary diseasefollowed by the reversion of liver fibrosis (Parsons et al., Healing the primary disease represents the ideal2004). Subsequently, fibrill ar collagen partial therapeutic measure in preventing liver fibrosisdegradation is initiated. Therefore, ECM production by development. Antiviral treatment associated with chronicactivated HSCs is altered, which leads to their apoptosis viral hepatitis may lead to viral clearance or suppression,(Elsharkawy et al., 2005). being followed by a reduction in fibrosis and portal

In vivo and in vitro studies have demonstrated NF-kB pressure (Roberts et al., 2007).role in blocking activated HSCs apoptosis, through a Another therapeutic measure regards stopping alcoholmechanism involving JNK cascade inhibition and AP-1 abuse in patients with alcoholic liver disease, orpathway (Czaja, 2003)responsible for modulating removing iron or copper excess in patients withcellular apoptosis (Oakley et al., 2005). Antagonists of precirrhotic genetic disease, hemochromatosis andNF-kB activity may represent a therapeutic target in Wilson's disease. A study conducted on a group ofreducing liver fibrosis, by stimulating HSCs apoptosis. patients with nonalcoholic steatohepatitis (NASH)

emphasized a correlation between weight loss and areduced fibrogenesis (Dixon et al., 2004).



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Inhibiting HSCs activation Leptin maintain HSCs in active state by stimulating

A therapeutic target in fibrotic reaction associated proliferation and inhibiting apoptosis via extracellular

with chronic hepatic disease is represented by inhibiting pathways involving ERK and protein kinase B

quiescent HSCs transdifferentiation in activated phosphorylation. Several studies on animal models have

myofibroblasts. In this direction, a reduction of oxidative reported a decrease in liver fibrosis in animals with leptinstress may represent a possible therapeutic alternative. deficiency (Saxena et al., 2004).

Thus, a series of antioxidants like vitamin E (Brown et al., Adiponectin is a proteic hormone expressed by

1997) , silimarinum (Pietrangelo et al., 1995), adipocytes whose seric levels are reduced in obese

phosphatidylcholin and S-adenosyl-L-methionine inhibit patients, leading to insulin resistance development.

HSCs activation, thus playing an antiapoptotic role for Adiponectin may counteract insulin resistance by TNF-

hepatocytes and therefore reducing liver fibrosis antagonism and the reduction of serum glucose and

progression. (Kawada et al., 1998). triglycerides levels (Balmer et al., 2010). Adiponectin

An experimental study conducted on an animal model activation of AMPK disrupts leptin-mediated hepatic

with liver cirrhosis induced by common bile duct fibrosis via suppressors of cytokine signalling SOCS-3

ligation, reported an improvement of liver fibrosis after (Handy et al., 2010). Chen et al. have demonstrated on

IGF-1 administration an important modulator of animal models that adiponectin administration leads to an

intermediary metabolism with role in oxidative stress improvement of alcoholic-induced hepatic injuries, byreduction (Canturk et al., 2003). reducing lipid droplets present in hepatocytes after

Another method of inhibiting HSCs activation alcohol abuse (Chen et al., 2007). Antifibrotic therapy

implies the administration of interferon-, whose benefic associated to liver disease may target these pathogenic

effects in ameliorating liver fibrosis were observed in pathways.

patients with chronic hepatic viral disease (Rockey et al., The association between pegylated interferon and

1994). ribavirin has led to a reduction of liver fibrosis

Therapeutic administration of HGF (Kim et al., 2005) progression (Poynard et al., 2002) in patients with

and IGF (Sanz et al., 2005) has determined an inhibition chronic viral hepatitis, by blocking viral replication and

in myofibroblasts' activation, which led to their apoptosis stopping hepatic injury (Shiratori et al., 2000).

HGF generates an antifibrotic effect via transcriptional Experimental studies have demonstrated that treatment

Ets-1 factor, which reduces HSCs TGF- synthesis and with -interferon diminishes liver fibrosis in patients with

stimulates MMP-1 synthesis (Ozaki et al., 2002). biliary fibrosis, thus demonstrating the existence of anA recent study has identified a new mechanism antifibrotic effect associated to the antiviral one. Non the

through which HGF reduces type I collagen synthesis in less, the antifibrotic effect of interferon is present only in

activated HSCs, by blocking TGF-mediated activation patients with viral eradication (Shiratori et al., 2000). In

pathways. HGF inhibits the transduction of TGF- vitro and in vivo experiments with corticosteroids have

profibrogenic signal, by increasing the interactions demonstrated an inhibitory effect on HSCs activating

between Gal-7 and phosphorylated Smad3. This leads to signalling molecules. In the past decades, corticosteroid

p-Smad3 blockage in cellular cytoplasm, followed by therapy was successfully used to diminish fibrosis

type I collagen synthesis inhibition (Inagaki et al., 2008). associated with autoimmune hepatitis (Czaja et al.,

Thiazolidinediones are synthetic ligands of PPAR- 2004).

(Galli et al., 2002) nuclear receptors identified on HSCs Raetsch et al. have reported a reduction of liver

membrane (Miyahara et al., 2000). They form a class of fibrosis by antagonising with pentoxiphylline the effect

antidiabetic drugs with benefic effects in liver disease by ge ne ra te d by TNF- (Rae ts ch et al ., 2002 ).

reducing HSCs activation (Marra et al., 2000). Pentoxiphylline is a metilxantine derivate which reduces

Leptin and adiponectin induce insulin resistance, activated HSCs synthesis of type I collagen, via

which intervenes in non-alcoholic liver diseases and inhibiting I kappa b degrading, which in turn blocks

chronic viral hepatitis pathogenesis and progression. NF-kB activation (Hernndez et al., 2007).

Leptin is an adipocytokine with profibrogenic Renin-angiotensin system amplifies inflammation by

properties by stimulating 2 (I) collagen synthesis generating oxidative stress. The therapies with inhibitors

(Bertolaniet al., 2008), whose excessive accumulation of the renin-angiotensin system and with antagonists of

represents the main feature of liver fibrosis (Saxena et type I receptor of type II angiotensinogen were

al.,2003; Ikejima et al., 2002) On the other hand, leptin successfully used in diminishing inflammation, which

inhibits MMP-1 synthesis and activity and it stimulates led to an improvement of liver fibrosis (Kurikawa et al.,

TIMP-1 expression (Lin et al., 2006). 2003).

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Inducing activated HSCs apoptosis HSCs, after FXR ligands stimulation of SHP identified

Activated HSCs apoptosis is the main mechanism in on activated HSCs membrane (Fiorucci et al., 2004).

reducing their number during the cure of liver injury Serial binding of FXR and SHP receptors inhibits TIMP-

(Iredale et al., 1998). Multiple apoptosis mediators, 1 expression in activated HSCs, after SHP's interaction

especially Fas/LasL, TNF and Bcl/Bax receptors, were with profibrogenic transcription factor JunD, resulting in

identified on activated HSCs membrane, therefore a new preventing JunD's binding with TIMP-1 promoter. At theantifibrogenic target may be represented by the same time, MMP-2 activity is doubled (Fiorucci et al.,

stimulation of these receptors which induce HSCs 2005). TIM P-1 inhibi tion is essent ia l: fi rst ,

apoptosis (Fallowfield et al., 2004). metalloproteases activity is no longer blocked, and

Both experimental and clinic evidence have shown second, HSCs no longer receive TIMP-1's antiapoptotic

the potential reversibility of liver fibrosis and cirrhosis by signals. Animal model studies have reported an increase

initiating activated HSCs apoptosis. The effects are thus synthesis of PPAR in activated HSCs, after the

targeted to the main cell involved in ECM production and stimulation of FXR ligands. PPAR agonists and FXR

in MMPs inhibition by TIMPs expression. ligands exert a synergic effect in reducing type I collagen

The progressive accumulation of ECM together with (Fiorucci et al., 2005). The combined effect of FXR

an impairment in the mechanism regulating its normal ligands and PPAR agonists therapy diminishes side

degradation, are responsible for liver fibrosis effects generated by the single use of PPAR agonists.

progression. MMP-1 is the main protease degrading type Natural killer cells are a major component of innateI collagen, the main collagen form present in fibrotic liver immunity. They play a role in reducing liver fibrosis

(Milani et al., 1994). MMPs inactivation is accomplished mainly by neutralising activated HSCs (Radaeva et al.,

by interaction with TIMPs molecules (Iredale, 2001). 2006; Melhem et al., 2006), and also by releasing two

Activated HSCs excessively synthesize TIMP-1 and proinflammatory cytokines INF and INF (Rockey et al.,

TIMP-250 which inhibit interstitial collagenases, leading 1994; Inagaki et al., 2003). NK cells are less efficient in

to a reduction in ECM degradation and consequently its alcohol abusing patients, hence the progression of liver

accumulation. TIMP-1 has an important role in activated fibrosis is accelerated in their case (Jeong et al., 2008).

HSCs survival by directly inhibiting their apoptosis. Its Experimental studies conducted on rats have reported

effect is dependent on MMPs inhibition (Murphy et al., that gliotoxin, a fungic metabolite, induces HSCs

2002). apoptosis in the absence of oxidative stress, by releasing

An important therapeutic antifibrotic method may be mitochondrial C cytochrome and by caspase-3 activation

represented by using TIMP antagonists to inhibit type I and ATP depletion (Anselmi et al., 2007).

collagen synthesis and to induce activated HSCs Bortesomib, a protease inhibitor, induces HSCsapoptosis (Parsons et al., 2004). apoptosis by blocking NF-kB activity, and by increasing

Hepatocytes' apoptosis exerts a profibrogenic the half-time of its inhibitors (Anan et al., 2006).

inflammatory signalling (Canbay et al., 2005). Caspases Cytokine therapy may be another therapeutic target

are a cistein protease family with multiple major roles in in preventing liver fibrosis. Gene therapies with growth

cellular apoptosis and necrosis. Canbay et al. have factors led to a reduction on liver fibrosis. An animal

experimentally emphasized on animal models that model study has observed a reduced proliferation and

caspase inhibitor IDN-6556 attenuates hepatic injury and apoptosis initiation in -SMA positive hepatic cells,

implicitly reducing fibrosis (Canbay et al., 2004). A following HGF administration (Kim et al., 2005).

major issue in using this therapy is represented by the

inhibitory effects on the apoptosis of cells which may Stimulation and increasing ECM degradation

have suffered DNA mutations, potentially leading to an Normally, quiescent HSCs produce type III IV

increased risk of HCC development. collagen, and small quantities of type I collagen (Milani

Farnesoid X receptor (FXR), a member of nuclear et al., 1989). During liver fibrogenesis, activated HSCs

transcription factor receptors family, is activated after become the main cells which increase the accumulation

binding with biliary acids, especially chenodeoxicholic of ECM, by an increased production of type I collagen

acid (CDCA). FXR controls genes which modulate bile (Milani et al., 1989; Maher et al., 1990).

flux and secretion (Rader et al., 2007). After identifying A major role in antifibrotic therapies is represented by

FXR on activated HSCs membrane, its involvement in existing ECM resorption. TGF- antagonists primary

cellular activity inhibition was also determined. A reduce ECM synthesis after the inhibition of activated

significant antifibrotic effect was generated HSCs, and secondly promotes ECM degradation via

experimentally in animal models, by using CDCA - the modulating TIMP's expression and by increasing

endogen ligand of FXR. interstitial collagenases activity. An in vivo study which

NR0B2, a small heterodimers partner (SHP), is a used gene therapy with urokinase-type plasminogen

member of intracellular nuclear transcription factor activator reported a reduction in collagen synthesis by

receptor family. In vivo and in vitro studies have reported modulating MMP, HGF and VEGF mediated signallinga reduction in type I collagen synthesis by activated pathways (Bueno et al., 2006).



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In primary bil iary cir rhosis , administrating protease inhibitors, determine both the inhibition of ECMursodeoxycholic acid generated an anti-inflammatory production and also the acceleration of its degradation. Aeffect, followed by a reduction of liver fibrosis (Degott et soluble recombined protease inhibitor mannose 6-al., 1999). A similarly result was experimentally phosphate (M-6-P) is in direct competition with TGF indemonstrated on an animal model, using a nitric oxide- binging on M-6-P receptor located on HSCs membrane,

releasing derivative of ursodeoxycholic acid that stopping their activation (de Bleser et al., 1995). Longselectively releases nitric oxide in the liver, which term administration of TGF- antagonists in humans maydecreased portal pressure (Fiorucci et al., 2003). Another lead to HCC development, by altering inflammatory andstudy on mice which used FXR receptors ligands has immune response, with a loss in growth inhibitionreported a increased choleresis and an antifibrotic effect mediated by TGF- (Matsuzaki et al., 2000).(Fiorucci et al., 2004). Renin-angiotensin system inhibitors are commonly

A series of studies have demonstrated the importance used as antifibrotic agents in chronic kidney and heartof TGF- and PDGF in mediating signalling pathways disease, especially by having reduced adverse effects inspecific for initiating liver fibrosis and tumorogenesis long term administration. A study conducted on two(Liu et al., 2006). These studies have emphasized that patient groups with chronic viral hepatitis C and non-genetic alterations in TGF- mediated signalling pathways alcoholic steatohepatitis have emphasized the beneficthrough Smad7 antagonist lead to a reduction of liver effects generated by renin-angiotensin inhibitors infibrosis and tumoral progression, after interacting with preventing liver fibrosis development (Yokohama et al.,

HSCs receptors (Mikula et al., 2006). 2004).Another method to inhibit liver fibrogenesis is based Son et al. have reported a diminution in activated

on administrating a powerful tyrosine kinase inhibitor HSCs proliferation, migration, collagen synthesis, asPTK/ZK, which blocks PDGF and TGF- mediated well as a reduced activity of additional profibrogenicsignalling pathways in activated HSCs (Liu et al., 2009). constituent genes, after stimulation with an adenovirus

The wnt pathway, another signalling pathway which which encodes a negative form dominated by PI3K,mediates renal and pulmonary fibrosis, may also play a controlled by an -SMA promoter. This adenovirus alsorole in initiating liver fibrosis. Some authors have induces cellular apoptosis (Son et al., 2009).reported a reduction in liver fibrosis by blocking this Cho et al. have emphasized the benefic effect ofsignalling pathway with Dickkopf-1 (DKK-1) an vasodilatatory substances like prostaglandine E2 and NOantagonist of Wnt coreceptor (Cheng et al., 2007). in antifibrotic therapies. These act by blocking

Several studies concentrated on understanding endotheline A receptors, thus stopping collagen synthesistranscriptional regulation have emphasized the and accumulation which leads to a diminishing in liverpossibility of inhibiting activated HSCs by blocking the fibrosis (Cho et al., 2000).activity of histone deacetylase (HDACs) an enzyme Liver fibrogenesis is inhibited by small molecularwhich plays a major role in chromatin alteration during weight molecules which block intracellular signallinggenetic transcription. Inhibitors with high HDACs pathways. These molecules represent a selectivespecificity may offer a selective method of blocking inhibitor of Rho mediated focal adhesion (Tada et al.,HSCs activation (Niki et al., 1999). 2001) and also an antisense for PDGF B-chain, with a

benefic effect in preventing experimental liver fibrosis(Borkham-Kamphorst et al., 2004).Neutralizing HSCs proliferative, profibrogenic,

HSP 47 is a collagen chaperone which is found incontractile and proinflammatory responseactivated HSCs, whose concentration in endoplasmicAnother target for antifibrotic therapies is blockingreticulum (Nagata, 1998) is correlated with an increasedproliferative, profibrogenic phases and contractileproduction of 1 (I) and 1 (III) collagen (Masuda et al.,response of activated HSCs, using antagonists of1994). Collagen synthesis in activated HSCs may becytokine receptors. Thus, there have been identifiedblocked by administration of liposomes containinginhibitors of -linoleic acid, lipooxigenase (Beno et al.,

vitamin A and siRNA which exerts inhibitory effects on1995) and for PPAR , PDGF and FGF receptors (Galli etHSP 47 (Sato et al., 2008).al., 2000).

Rapamycin an immunosupressor administrated afterSeveral researchers have reported a reduction ofliver transplant may represent a new therapeutic agentactivated TGF-b which leads to a diminishing in liverwhich inhibits HSCs proliferation (Zhu et al., 1999). Itsfibrosis progression in rats, after administrating camostatlong term administration increases the risk to developmesilate (Okuno et al., 2001). Yoshiji et al. havehepatic artery thrombosis (Trotter, 2003).demonstrated, in a study conducted on rats, that imatinib

Bennett et al. have observed in vivo and in vitro amesylate an inhibitor of tyrosine kinase receptorreduction in collagen synthesis by activated HSCs,determines a diminution of liver fibrosis due to aassociated with an increase in ECM degradation, aftersignificant reduction of activated HSCs proliferation andadministrating relaxin a natural peptide hormone, whosemigration, induced by PDGF-BB, as well as areceptors are present on activated HSCs membranediminishing in -SMA and mRNA 2 (I) procollagen(Bennett et al., 2005).expression in activated HSCs (Yoshiji et al., 2005).

TGF-antagonists, like monoclonal antibodies and

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