Hepatic fibrosis and cirrhosis The (myo)fibroblastic cell subpopulations involved

The International Journal of Biochemistry&Cell Biology38(2006)135–151Medicine in focusHepaticfibrosis and cirrhosis:The(myo)fibroblastic cellsubpopulations involvedChristelle Guyot a,S´e bastien Lepreux a,b,c,Chantal Combe a,Evelyne Doudnikoff c, Paulette Bioulac-Sage a,b,Charles Balabaud a,Alexis Desmouli`e re a,∗a GREF,INSERM E0362,Universit´e Victor Segalen Bordeaux2,146,rue L´e o-Saignat,Bordeaux Cedex,F-33076Franceb CHU Bordeaux,Hˆo pital Pellegrin,Service d’Anatomie Pathologique,Bordeaux,F-33076Francec Laboratoire d’Histologie-Embryologie,UFR II,Universit´e Victor Segalen Bordeaux2,Bordeaux,F-33076FranceReceived9June2005;received in revised form28August2005;accepted31August2005AbstractFibrosis,defined as the excessive deposition of extracellular matrix in an organ,is the main complication of chronic liver damage. Its endpoint is cirrhosis,which is responsible for significant morbidity and mortality.The accumulation of extracellular matrix observed infibrosis and cirrhosis is due to the activation offibroblasts,which acquire a myofibroblastic phenotype.Myofibroblasts are absent from normal liver.They are produced by the activation of precursor cells,such as hepatic stellate cells and portal fibroblasts.Thesefibrogenic cells are distributed differently in the hepatic lobule:the hepatic stellate cells resemble pericytes and are located along the sinusoids,in the Disse space between the endothelium and the hepatocytes,whereas the portalfibroblasts are embedded in the portal tract connective tissue around portal structures(vessels and biliary structures).Differences have been reported between these twofibrogenic cell populations,in the mechanisms leading to myofibroblastic differentiation,activation and “deactivation”,but confirmation is required.Second-layer cells surrounding centrolobular veins,fibroblasts present in the Glisson capsule surrounding the liver,and vascular smooth muscle cells may also express a myofibroblastic phenotype and may be involved infibrogenesis.It is now widely accepted that the various types of lesion(e.g.,lesions caused by alcohol abuse and viral hepatitis) leading to liverfibrosis involve specificfibrogenic cell subpopulations.The biological and biochemical characterisation of these cells is thus essential if we are to understand the mechanisms underlying the progressive development of excessive scarring in the liver. These cells also differ in proliferative and apoptotic capacity,at least in vitro.All this information is required for the development of treatments specifically and efficiently targeting the cells responsible for the development offibrosis/cirrhosis.©2005Elsevier Ltd.All rights reserved.Keywords:Fibroblast;Myofibroblast;Extracellular matrix;Liverfibrosis;Cirrhosis;RemodellingAbbreviations:CCl4,carbon tetrachloride;CRBP-1,cellular retinol-binding protein-1;MMP,matrix metalloproteinase;TGF-␤1,transforming growth factor-␤1;TIMP-1,tissue inhibitor of metalloproteinase-1∗Corresponding author.Tel.:+33557571771;fax:+33556514077.E-mail address:Alexis.Desmouliere@gref.u-bordeaux2.fr (A.Desmouli`e re).1.IntroductionThe processes of liver repair and offibrogenesis resemble a wound healing process.When injury and the associated acute inflammation response result in moder-ate cell necrosis and extracellular matrix damage,tissue repair normally takes place.In this process,dead cells are replaced by normal tissue,with regeneration of spe-cialised cells by proliferation of the surviving ones,for-mation of a granulation tissue,and tissue remodelling1357-2725/$–see front matter©2005Elsevier Ltd.All rights reserved. doi:10.1016/j.biocel.2005.08.021136 C.Guyot et al./The International Journal of Biochemistry&Cell Biology38(2006)135–151with scar formation.The specific regenerative capacities of the liver generally allow it to reconstitute itself entirely following acute,moderate lesions.However,chronic injuries to the liver do not always heal as effectively and fibrosis is the main complication of the many known chronic liver diseases.Various types of chronic injury, due to alcohol abuse,viral hepatitis(especially Hep-atitis B and C),drugs,metabolic diseases(mainly due to overload of iron or copper),autoimmune destruction of hepatocytes or bile duct epithelium,and congenital abnormalities may lead to liverfibrosis(for review,see Bataller&Brenner,2005).The liver parenchyma is divided into functional units called lobules.The lobules are polygonal,generally hexagonal,and each is1–2mm in diameter and com-posed of a labyrinth of interconnected hepatocyte plates separated by endothelium-lined sinusoids.Each lobule is crossed by a central structure,the centrolobular vein. The hepatocyte plates radiate out from the centrolobu-lar vein to the perimeter of the lobule;the portal triads (portal vein,hepatic artery,and bile ductule),and the surrounding connective tissue are typically found at the angles of the polygon(Fig.1a and b).Liverfibrosis is defined as the abnormal accumu-lation of extracellular matrix in the liver.Its endpoint is cirrhosis,which is responsible for a significant mor-bidity and mortality.Cirrhosis is an advanced stage of fibrosis,characterised by the formation of regenerative nodules of liver parenchyma separated byfibrotic septa. Three major mechanisms are involved in the generation of cirrhosis:cell death,aberrant extracellular matrix deposition(fibrosis),and vascular reorganisation. Fibrous septa connecting the portal tracts and hepatic veins form,leading to portovenous and arteriovenous shunting,and effective bypassing of the parenchymal nodules.This results in vascular thrombosis of the medium-sized and large portal veins and of the hepatic veins and the progression of parenchymal extinction to full-blown cirrhosis.Parenchymal extinction is the loss of continuous hepatocyte layers due tofibrosis of the parenchymal stroma(Wanless,2002).In most cases, significant lesions are observed only after months or years of injury.However,they may appear more rapidly in congenital liver diseases,such as biliary atresia.Liver fibrosis is reversible,whereas cirrhosis is generally irre-versible(Benyon&Iredale,2000;Bioulac-Sage et al., 2000).Prevention of the progression offibrosis to cir-rhosis is therefore a major clinical goal.Unfortunately, current treatments of the underlying diseases responsible for liver damage are only partly successful in preventing this progression.The poor prognosis of cirrhosis is aggravated by the frequent occurrence of hepatocellular carcinoma,which may also develop,albeit much more rarely,in normal or only slightlyfibrous livers.The accumulation of extracellular matrix observed infibrosis and cirrhosis is due to the activation of fibroblasts,which acquire a myofibroblastic phenotype. Myofibroblasts are absent from normal liver.They are produced by the activation of precursor cells,such as hepatic stellate cells(for review,see Lotersztajn,Julien, Teixeira-Clerc,Grenard,&Mallat,2005).It has been suggested that liverfibrogenic cells are heterogeneous, as the portalfibroblasts present in portal tracts may also play a major role in liverfibrogenesis(Dranoff et al.,2002;Kinnman et al.,2003;Knittel et al.,1999b; Tang,Tanaka,Marumo,&Sato,1994;Tuchweber, Desmouli`e re,Bochaton-Piallat,Rubbia-Brandt,& Gabbiani,1996).In this review,we will identify the variousfibrogenic cell subpopulations involved in liver fibrogenesis,and discuss the mechanisms underlying myofibroblastic differentiation and extracellular matrix deposition in various liver disease.Finally,we will eval-uate the possibility of tissue remodelling,which may renderfibrosis and cirrhosis reversible in some cases.2.Definition of the myofibroblastInflammation occurs in response to tissue damage, with the formation of a provisional matrix favouring cell migration and proliferation in the lesion.Granulation tissue,facilitating the replacement of the injured tissue, then develops.This tissue displaysfibroblast prolifera-tion,angiogenesis,and extracellular matrix deposition. During tissue repair and granulation tissue formation,fibroblasts acquire the smooth muscle features char-acteristic of myofibroblasts(for review,see Serini& Gabbiani,1999;Tomasek,Gabbiani,Hinz,Chaponnier, &Brown,2002),the main cell type in granulation tissue. Myofibroblasts therefore appear to be a morphological and functional intermediate betweenfibroblasts and the smooth muscle cells.Myofibroblasts contain cytoplas-mic bundles of microfilaments or stressfibres,which play a role in contraction,via mechanisms similar but not identical to those in smooth muscle cells.Myofi-broblasts are surrounded by an irregular basal mem-brane,and are connected to each other by gap junctions and to the extracellular matrix by focal contacts called fibronexi—transmembrane complexes containing intra-cellular microfilaments in continuity with extracellular fibronectinfibrils(Eyden,1993).Myofibroblasts are the main cellular type involved in extracellular matrix depo-sition during tissue repair,but they are also responsible for synthesising enzymes involved in matrix degrada-tion,tissue remodelling,and scar formation.C.Guyot et al./The International Journal of Biochemistry&Cell Biology38(2006)135–151137Fig.1.(a)Normal liver.Tissue section of a formalin-fixed paraffin-embedded normal human liver(haematoxylin-eosin-saffranin staining)showing a centrolobular vein(CLV),and a portal zone with a portal vein(PV),arteries(A),and bile ducts(BD),surrounded by dense connective tissue containing collagenous extracellular matrix and portalfibroblasts.In the parenchyma,numerous hepatocytes are seen,but the sinusoidal structures are not visible due to tissue treatment.(b)Diagram of a lobule.This schematic diagram shows the three-dimensional structures of the hepatic lobule as revealed by scanning electron microscopy.The central structure in the lobule is the centrolobular vein.Hepatocytic plates radiate out from the centrolobular vein to the perimeter of the lobule;the portal triads(portal vein,hepatic artery,and bile ductule),and the surrounding connective tissue are typically found at the angles of the polygon(for details,see text).CLV,centrolobular vein;K,Kupffer cell;HSC,hepatic stellate cells;HP, hepatocyte plate;SE,sinusoidal endothelium;S,sinusoid;BC,bile canaliculus;A,hepatic artery;CH,canal of Hering;PV,portal vein;BD,bile ductule;LmP,limiting plate.(c)Schematic diagram of the various liverfibroblastic cells able to acquire a myofibroblastic phenotype and involved infibrogenesis.The portalfibroblasts(PF)located in the portal tract connective tissue around bile ducts(BD),portal arteries(A),and portal veins (PV),and the second-layer cells(SLC),fibroblasts located around the smooth muscle cells(SMC)and the endothelium(E)of the centrolobular veins(CLV),can acquire a myofibroblastic phenotype,and these cells do not seem to be able to reacquire a quiescent phenotype;in contrast,the hepatic stellate cells(HSC)containing lipids droplets and located in the Disse space between the hepatocytes(H)and the sinusoidal endothelium (SE)can modulate their myofibroblastic differentiation,and present pericyte-like features suggesting that they function as liver-specific pericytes participating in the regulation of sinusoidal blood pressure.Myofibroblasts(MF)present microfilaments bundles and secrete large amounts of extracellular matrix.138 C.Guyot et al./The International Journal of Biochemistry&Cell Biology38(2006)135–151Myofibroblasts express different sets of cytoskeletalproteins that can be used as markers of cell differ-entiation;the study of their expression allows thecharacterization offibroblastic phenotypic modifica-tions corresponding to functional changes occurringduring physiological and pathological repair(for review,see Desmouli`e re&Gabbiani,1996;Schmitt-Gr¨a ff,Desmouli`e re,&Gabbiani,1994).The cytoskeleton of eukaryotic cells consists of threetypes offilament:(i)intermediatefilaments,whichstrengthen cells against mechanical stress;their compo-sition depends on cell type,(ii)microtubules,which playa crucial role in cell organisation,and direct intracellulartransport,and(iii)actin microfilaments,which deter-mine the shape of the cell surface and are necessary forwhole-cell locomotion.Actin microfilaments are thinandflexible.Six isoforms of actin exist:two cytoplasmicor non-muscle actins(␤and␥),and four muscle actins(two striated muscle actins,␣-skeletal and␣-cardiac,and two smooth muscle actins,␣and␥).Actinfilamentsassociate with many actin-binding proteins(capping,severing,crosslinking,bundling,nucleating,sequester-ing,or motor proteins),allowing thefilament to performvarious functions.Cytoskeleton components,and actinisoforms in particular,are useful tools for evaluatingphenotypic modifications in response to physiological orpathological situations(Chaponnier&Gabbiani,2004).Fully differentiated myofibroblasts express␣-smoothmuscle actin(Darby,Skalli,&Gabbiani,1990).How-ever,as pointed out by Tomasek et al.(2002),fibroblasticcells may present in some situations morphologicalcharacteristics of myofibroblasts(e.g.,microfilamentbundles of cytoplasmic actins),but they do not express ␣-smooth muscle actin.The term“protomyofibroblasts”has been proposed for these cells(Tomasek et al.,2002).Fibroblasts cultured in the presence of foetal calf serumgenerally display a mixture of protomyofibroblastic andmore differentiated myofibroblastic phenotypes.Thepresence of␣-smooth muscle actin is directly related tothe contractile activity of myofibroblasts.Indeed,it hasrecently been demonstrated,both in vitro and in vivo,that␣-smooth muscle actin levels are directly correlatedwith myofibroblast contraction(Hinz,Celetta,Tomasek,Gabbiani,&Chaponnier,2001a;Hinz,Mastrangelo,Iselin,Chaponnier,&Gabbiani,2001b).Protomyofi-broblasts and myofibroblasts may also produce othersmooth muscle cytoskeletal proteins,including desmin,caldesmon,and smooth muscle-myosin heavy chains(for review,see Desmouli`e re&Gabbiani,1996).Inter-estingly,global expression profiling studies offibroblastresponses to transforming growth factor-␤1(TGF-␤1)–a major inducer of myofibroblastic differentiation (Desmouli`e re,Geinoz,Gabbiani,&Gabbiani,1993)–have demonstrated the up-regulation of a number of genes generally expressed by highly differentiated smooth muscle cells.These genes included those encoding smooth muscle-myosin heavy chains,basic calponin,and smoothelin(Chambers,Leoni,Kaminski, Laurent,&Heller,2003).The stimulation of collagen type I and of␣-smooth muscle actin synthesis by TGF-␤1is strictly dependent on cellularfibronectin, and the ED-A splice variant of this glycoprotein in particular(Serini et al.,1998).Thus,myofibroblast differentiation is a complex process,regulated by at least one cytokine,an extracellular matrix component and mechanical tension(Hinz&Gabbiani,2003).Recent studies taking thesefindings into account have com-pared thefibronexus to a mature or supermature focal contact—a three-dimensional transcellular structure containing thefibronectin isoform ED-A and␣-smooth muscle actin.These structures are organised by forces of intracellular and extracellular origin and are involved in establishment and modulation of the myofibroblastic phenotype(Dugina,Fontao,Chaponnier,Vasiliev,& Gabbiani,2001).It has also been shown that␣-smooth muscle actin is essential for focal adhesion matu-ration in myofibroblasts(Hinz,Dugina,Ballestrem, Wehrle-Haller,&Chaponnier,2003).Myofibroblasts, unlike quiescentfibroblasts,also develop cadherin-type cell–cell adherens junctions,which are instrumental in myofibroblast contractile activity(Hinz,Pittet, Smith-Clerc,Chaponnier,&Meister,2004).Typical myofibroblasts have been found in normal tis-sues in various organs(e.g.,uterine submucosa,intesti-nal pericryptal cells,testicular stroma,periodontal liga-ment;for review,see Sch¨u rch,Seemayer,&Gabbiani, 1998),in which these cells are involved in visceral con-traction and/or organ remodelling.We can identify three groups of pathological situations in which myofibrob-lasts are the predominant cell type:response to injury and repair phenomena(hypertrophic scars and burn contrac-ture,organfibrosis),quasineoplastic proliferative condi-tions(fibromatosis),and stromal response to neoplasia (for review,see Sch¨u rch et al.,1998).3.(Myo)fibroblastic cells involved in liverfibrosis 3.1.The hepatic stellate cellHepatic stellate cells,which account for about 5–8%of cells in the normal liver,are characterised by a perisinusoidal distribution in the Disse space and long processes extending along and around sinusoids, between the hepatocyte plates(Fig.1a and b).ElectronC.Guyot et al./The International Journal of Biochemistry&Cell Biology38(2006)135–151139microscopy has shown that the nucleus-to-nucleus distance between two adjacent hepatic stellate cells is∼40␮m.Eight to10hepatic stellate cells lie along each sinusoid,between the centrolobular vein and the portal tract.The relationship between hepatic stellate cells and sinusoidal tubes is one of the most important features of these cells.Indeed,the close association of hepatic stellate cells with endothelial cells resembles that of pericytes in capillaries.However,in normal liver, the endothelium is discontinuous and presents multiple fenestrations without diaphragms,allowing the rapid transport of solutes across the subendothelial space.In normal liver,a basal lamina-like substance separates the two cell types but there is no true basement membrane. Cytoplasmic lipid droplets containing Vitamin A are present in most(75%)hepatic stellate cells in normal liver(Bioulac-Sage,Lafon,Le Bail,&Balabaud,1988; Sztark et al.,1986),and hepatic stellate cells constitute the largest cellular reservoir of Vitamin A in the body. Hepatic stellate cells are activated and change phenotype under conditions of stress or injury,often losing their lipid droplets upon activation.The biological markers of these cells also seem to change according to activation level or position within the lobule,making it difficult to use these markers to follow physiological or patho-logical responses.On activation,the hepatic stellate cells acquire a myofibroblastic phenotype(Fig.1c), contributing to the excessive extracellular matrix deposition observed in the pathological conditions of fibrosis and cirrhosis.Capillarisation of the sinusoids also occurs,with a continuous endothelium formed,and the presence of a true basal lamina.The experimental model of carbon tetrachloride (CCl4)treatment in rats has been extensively used to study liverfibrogenesis.It is widely accepted that a toxic metabolite of this molecule causes liver damage,but the identity of this metabolite remains l4treat-ment induces the necrosis of hepatocytes around cen-trolobular veins,and the accumulation of inflammatory cells.Following a single acute treatment,tissue repair occurs,with the appearance of a moderate number of␣-smooth muscle actin-expressing myofibroblastic cells. Following chronic injury,a large number of myofibrob-lastic cells accumulate around centrolobular veins.Septa containing myofibroblastic cells expressing␣-smooth muscle actin then develop between centrolobular areas, and large amounts of extracellular matrix are deposited. It is generally thought that the activated hepatic stellate cells are responsible for this extracellular matrix accu-mulation(for review,see Reeves&Friedman,2002). Hepatic stellate cells have been shown to migrate in vitro (Marra et al.,1999),suggesting that they may migrate to the lesion and take part in the repair process(Kinnman et al.,2000).Lorena et al.(2004)recently reported thatfibrillin-1 and␣-smooth muscle actin were both limited to vessel walls in the normal rat liver.Fibrillin-rich microfibrils are important structural elements abundant in connec-tive tissues.Depending on the location,microfibrils and microfibril-associated glycoproteins may be associated with an amorphous elastin core to form elasticfibres. In contrast to what has been reported for human liver (Dubuisson et al.,2001),in rat liver,fibrillin-1was either absent or present in only very small quantities in the Disse space around the sinusoids.After CCl4treatment, double immunohistochemical staining demonstrated the presence of bothfibrillin-1and␣-smooth muscle actin infibrotic lesions.This suggests that␣-smooth mus-cle actin-positive myofibroblasts are responsible for thefibrillin-1deposition observed duringfibrogenesis. Elastin and␣-smooth muscle actin were co-localised in septa developing after CCl4treatment,but activated␣-smooth muscle actin-positive hepatic stellate cells in the parenchyma did not contain elastin.Thus,in the CCl4 model,at least for the studied time points,the typical acti-vated hepatic stellate cells containing␣-smooth muscle actin seem to play little or no part in elastin deposition. These observations suggest that different liverfibroblast subpopulations are involved in deposition of the different extracellular matrix components and/or that there are dif-ferent subpopulations of hepatic stellate cells.Indeed,it has long been suggested that several subclasses of hep-atic stellate cells are present in the parenchyma(for a recent review,see Friedman,2004).An elegant study in mice transgenic for two reporter genes(two differentflu-orescent protein reporter genes expressed under control of the mouse␣-smooth muscle actin and collagen␣1(I) promoter/enhancers)recently clearly demonstrated in primary cultures of hepatic stellate cells,heterogeneous gene expression in hepatic myofibroblasts(Magness, Bataller,Yang,&Brenner,2004).3.2.The portalfibroblastThe animal model of bile duct proliferation andfibro-sis after ligation of the common bile duct has attracted considerable attention,because it simulates many histological features of human chronic biliaryfibrosis and cirrhosis.Few studies have examined the responses of differentfibrogenic cells and the cellular origin of the extracellular matrix components in this model offibrosis. It has been suggested that the cells involved are hepatic stellate cells reaching the portal region by proliferation and migration(Kinnman et al.,2000,2001).However,140 C.Guyot et al./The International Journal of Biochemistry&Cell Biology38(2006)135–151the involvement of portal tractfibroblasts around biliary structures in the portalfibrosis induced by common bile duct ligation has now been clearly demonstrated,at least for the early stages(Kinnman et al.,2003;Tuchweber et al.,1996).Hepatic stellate cells may become involved later,when the growing portal areas invade the parenchyma(Kinnman&Housset,2002;Tuchweber et al.,1996).Electron micrographs of the portal area in normal rats have demonstrated the presence of portal fibroblasts in the connective tissue around vessels and biliary structures,with a typical appearance of quiescent fibroblasts presenting few cytoplasmic processes.Two days after ligation,manyfibroblasts are observed surrounding proliferating ductules,with a few of these fibroblasts showing microfilament bundles(a feature of myofibroblastic differentiation)in their cytoplasmic processes(Tuchweber et al.,1996).The proliferation of biliary structures induced by common bile duct ligation is accompanied by a proliferation of portalfibroblasts, which form onion-like structures around biliary struc-tures.These portalfibroblasts acquire a myofibroblastic phenotype and are involved in the early deposition of extracellular matrix in the portal zones(Desmouli`e re et al.,1997)(Fig.1c).The portal connective tissue of normal liver contains only quiescentfibroblasts,with no portal myofibroblasts.Portal myofibroblasts appear when a lesion occurs in the portal zone.The bile duct ligation model is characterised by moderate inflam-mation,and the main factors responsible for inducing portal lesions are an increase in biliary pressure(Slott, Liu,&Tavoloni,1990)and probable modifications in bile composition.Cytokines,such as platelet-derived growth factor(Kinnman et al.,2003),and connective tissue growth factor(Sedlaczek et al.,2001)are also involved.Finally,recent studies on animal material and human samples have suggested that mesenchymal cells in the portal tract are of crucial importance in the development of liverfibrosis and cirrhosis(for review, see Ramadori&Saile,2004a).Transmission electron microscopy of normal human liver has demonstrated the presence of numerousfibroblastic cells in the portal tracts,constituting a major reservoir offibrogenic cells (Fig.2).3.3.Otherfibrogenic cellsSecond-layer cells located around centrolobular veins and described by Bhunchet and Wake(1992)(Fig.1c),fibroblasts present in the Glisson capsule surrounding the liver,and vascular smooth muscle cells may also be involved infibrogenesis,probably by modulation to myofibroblasticcells.Fig. 2.Normal human liver(transmission electron microscopy). Around the biliary epithelial cells of a bile duct(BD),numerous por-talfibroblasts(arrows)are observed.These cells(portalfibroblasts), together with hepatic stellate cells,represent a reservoir offibroblastic cells potentially involved infibrogenesis.After portal tract injury,por-talfibroblasts can proliferate and acquire a myofibroblastic phenotype.Beside very commonly used experimental models such as CCl4treatment and bile duct ligation,fibrosis can also be induced by the intraperitoneal injection of pig serum in rats(Bhunchet&Wake,1992;Rubin,Hutterer, &Popper,1968).These pig serum-sensitised animals accumulate extracellular matrix in the centrolobular and periportal zones and no necrotic hepatocytes or acute inflammatory reactions are observed.Prominentfibrotic septa are observed,extending out in a radial pattern from central veins and portal zones,resulting in the forma-tion of pseudolobules.However,thefibroblastic cells involved and the mechanisms leading to hepaticfibro-sis in this model are unknown.The vascular smooth muscle cells residing in the wall of portal vein branches and portal arteries have been implicated in the typical portal/periportalfibrosis observed in chronic schistoso-miasis(Andrade,Guerret,&Fernandes,1999).In addition,myofibroblasts are a major component in the stroma reaction,which develops in hepatocel-lular carcinoma(for review,see Desmouli`e re,Guyot, &Gabbiani,2004).In this cancer,they are involved in the synthesis of stromal extracellular matrix com-ponents,extracellular matrix-degrading proteinases,and pro-invasive factors,including hepatocyte growth factor.A number of experimental models have been developed but the origin of the(myo)fibroblastic cells involved in the stroma reaction observed in hepatocellular car-cinoma remains unclear.Progenitorfibroblastic cells have never been detected in the liver.In contrast,in the dermis,progenitor cellsC.Guyot et al./The International Journal of Biochemistry&Cell Biology38(2006)135–151141located in the dermal sheath surrounding the hair fol-licle have been shown to maintain and regenerate the dermal papilla and to play an important role in repair-ing the dermis after injury(Jahoda&Reynolds,2001). It is thought that the canals of Hering,which have been demonstrated to proliferate in disease states,may har-bour liver stem cells(Fausto&Campbell,2003;Theise et al.,1999).This suggests that the canals of Hering and the surrounding stromal cells may constitute a niche for hepatic stem cells(Saxena&Theise,2004).The canals of Hering have a highly strategic distribution(Fig.1b): they begin in the lobules,are lined partly by bile duct epithelial cells and partly by hepatocytes,and conduct bile from the bile canaliculi to terminal bile ducts in the portal tracts(Roskams et al.,2004).The production of large numbers of(myo)fibroblasts by local epithelial-mesenchymal transition has been reported during renal fibrogenesis(for review,see Kalluri&Neilson,2003). Further studies are required to confirm this process in the kidney but no equivalent mechanism has ever been observed in the liver.In addition tofibrogenic cells resident in the liver, circulating cells may play an essential role infibroge-nesis.An analysis of the tissues of humans receiving sex-mismatched organ or bone marrow transplants iden-tified bone marrow-derived myofibroblasts participating infibrogenic reactions in the liver(Forbes et al.,2004). Evidence has also been obtained that circulating pre-cursor cells migrate into wounds and contribute to the (myo)fibroblastic population of the repair tissue(Abe, Donnelly,Peng,Bucala,&Metz,2001).Indeed,Bucala, Spiegel,Chesney,Hogan,and Cerami(1994)identified a novel blood-borne cell subpopulation withfibroblast-like properties,which they referred to asfibrocytes(for review,see Quan,Cowper,Wu,Bockenstedt,&Bucala, 2004).Peripheral bloodfibrocytes may rapidly enter the injury site at the same time as circulating inflammatory cells.For example,it has been suggested that circulating fibrocytes represent a major source offibroblasts during the healing of extensive burn wounds,in which it may be difficult forfibroblasts to migrate from the edges of the injury(Yang et al.,2002).However,although this process seems very likely,no role forfibrocytes in the develop-ment of liverfibrosis has yet been definitively demon-strated.In conclusion,all these processes(the involve-ment offibrocytes,a process of epithelial-mesenchymal transdifferentiation,or the presence of liver progenitor fibroblastic cells)may represent alternative sources of (myo)fibroblasts,and could be involved in specific situ-ations,particularly when the size of the lesion increases, and when recruitment of the localfibroblasts is unable to“comply with the request”.3.4.General considerationsThe data now available suggest that there is a need to reconsider the role of hepatic stellate cells in the devel-opment of liverfibrosis.The pericyte-like characteristics of hepatic stellate cells,including their close relationship with endothelial cells and their expression of smooth muscle markers,have been discussed extensively in pre-vious publications(Blomhoff&Wake,1991;Pinzani, 1995).Hepatic stellate cells are probably“resting peri-cytes”that may be rapidly activated(e.g.,in response to changes in blood pressure),acquiring contractile prop-erties enabling them to regulate local blood pressure. Hepatic stellate cells have been shown to be activated early in transplantation,as demonstrated by the acute, transient production of␣-smooth muscle actin(Rubbia-Brandt et al.,1997).Moreover,ex vivo liver perfusion induces:(1)the early activation of hepatic stellate cells, which begin to produce␣-smooth muscle actin and(2) significant changes in the perisinusoidal extracellular matrix(Costa et al.,2001).Thesefindings are consis-tent with the view that hepatic stellate cells function as liver-specific pericytes,participating in the regulation of sinusoidal blood pressure.Several groups have shown that activated hepatic stel-late cells can revert to quiescence(Gaca et al.,2003; Sohara,Znoyko,Levy,Trojanowska,&Reuben,2002), suggesting that hepatic stellate cells have a plastic phe-notype.The role played by the extracellular matrix envi-ronment has been highlighted,and it has been shown that a basement membrane-like matrix induces the deac-tivation of hepatic stellate cells(Gaca et al.,2003).It has also clearly been demonstrated that the stiffness of the surrounding environment determines the phenotype of hepatic stellate cell(Wells,2005).Many studies have shown that activated hepatic stellate cells proliferate(for review,see Friedman,2000).Portal(myo)fibroblasts can also undergo several subcultures.It has been suggested that pure populations of hepatic stellate cells do not pro-liferate(Knittel et al.,1999b;Ramadori&Saile,2004a; personal observations),but further studies are required to confirm this.Rat liver myofibroblasts and hepatic stellate cells differ in CD95-mediated apoptosis and response to tumour necrosis factor-␣(Saile et al.,2002).Insulin-like growth factor-1induces DNA synthesis and apoptosis in rat liver hepatic stellate cells but DNA synthesis and pro-liferation in rat liver myofibroblasts(Saile et al.,2004). It has been suggested that liverfibroblastic cells other than typical hepatic stellate cells(e.g.,portalfibroblasts) may have been analysed in studies of hepatic stellate cells after several passages,and this interpretation has clearly been accepted by the authors of several recent。