hepatic stellate cells and liver fibrosis · hepatic stellate cells and liver fibrosis...

Hepatic Stellate Cells and Liver FibrosisJuan E. Puche,1,2 Yedidya Saiman,1 and Scott L. Friedman*1

ABSTRACTHepatic stellate cells are resident perisinusoidal cells distributed throughout the liver, with aremarkable range of functions in normal and injured liver. Derived embryologically from sep-tum transversum mesenchyme, their precursors include submesothelial cells that invade the liverparenchyma from the hepatic capsule. In normal adult liver, their most characteristic feature isthe presence of cytoplasmic perinuclear droplets that are laden with retinyl (vitamin A) esters.Normal stellate cells display several patterns of intermediate filaments expression (e.g., desmin,vimentin, and/or glial fibrillary acidic protein) suggesting that there are subpopulations withinthis parental cell type. In the normal liver, stellate cells participate in retinoid storage, vasoregu-lation through endothelial cell interactions, extracellular matrix homeostasis, drug detoxification,immunotolerance, and possibly the preservation of hepatocyte mass through secretion of mi-togens including hepatocyte growth factor. During liver injury, stellate cells activate into alphasmooth muscle actin-expressing contractile myofibroblasts, which contribute to vascular distor-tion and increased vascular resistance, thereby promoting portal hypertension. Other features ofstellate cell activation include mitogen-mediated proliferation, increased fibrogenesis driven byconnective tissue growth factor, and transforming growth factor beta 1, amplified inflammationand immunoregulation, and altered matrix degradation. Evolving areas of interest in stellate cellbiology seek to understand mechanisms of their clearance during fibrosis resolution by eitherapoptosis, senescence, or reversion, and their contribution to hepatic stem cell amplification, re-generation, and hepatocellular cancer. C© 2013 American Physiological Society. Compr Physiol3:1473-1492, 2013.

IntroductionLiver fibrosis is a common outcome of virtually all chronichepatic insults including viral hepatitis (i.e., hepatitis Band C), alcoholic or obesity-associated steatohepatitis (i.e.,nonalcoholic steatohepatitis (NASH)), parasitic disease(i.e., schistosomiasis), metabolic disorders (i.e., Wilson’s),hemochromatosis and other storage diseases, congenitalabnormalities, autoimmune and chronic inflammatory condi-tions (i.e., sarcoidosis), and drug toxicity, among others (102).

Fibrosis was described in 1951 as a passive process with“no direct evidence of fibrous tissue proliferation but the sug-gestion of connective tissue appearance just by stromal con-densation due to hepatocyte cell collapse.” However, in 1978,a growing body of evidence prompted the World Health Orga-nization to update its definition of fibrosis as “the presence ofexcess collagen due to new fiber formation (3).” It is virtuallyaxiomatic now that liver fibrosis results from enhanced pro-duction of extracellular matrix (ECM) due to accumulationand activation of myofibroblasts in the context of ongoingor repetitive liver damage. Early studies focused on meth-ods to isolate and grow primary hepatic stellate cells (HSCs)in culture establishing them as one of the main sources ofmyofibroblasts in liver parenchymal disease resulting fromhepatocyte as oppsed to biliary injury (49, 71). Immediatelythereafter, the concept of stelllate cell “activation” emerged,which represents a transdifferentiation of the cell during liverinjury from a quiescent state that is rich in vitamin A droplets,

to one that is proliferative fibrogenic and contractile (62, 82,83, 234, 241, 289). While HSCs are a major source of myofi-broblasts, mounting evidence also implicates portal fibrob-lasts as a source when the injury is to bile ducts rather thanhepatocytes (53, 162).

From early studies focusing on the role of the stellatecells solely as a source of ECM during liver injury, a sus-tained effort has subsequently uncovered broadening rolesof the cell type as a source of regenerative cytokines, animmunomodulatory cell with a range of activities and onethat serves roles far beyond those envisioned at the time of itsisolation 35 years ago (65). From these studies realistic hopeshave emerged for exploiting features of stellate cell activationand hepatic inflammation in devising effective antifibrotic andregenerative therapies for patients with chronic liver diseaseand fibrosis.

This article builds upon a comprehensive review of stel-late cell biology in 2008 by one of the authors (S.L.F.) (65),

*Correspondence to [email protected] of Liver Diseases, Icahn School of Medicine at Mount SinaiHospital, New York, New York2University CEU-San Pablo, School of Medicine, Institute of AppliedMolecular Medicine (IMMA), Madrid, SpainPublished online, October 2013 (comprehensivephysiology.com)DOI: 10.1002/cphy.c120035Copyright C© American Physiological Society.

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Hepatic Stellate Cells and Liver Fibrosis Comprehensive Physiology

Figure 1 Appearance of hepatic stellate cells and the sinusoidal microenvironment in normal and injuredliver. In normal liver, stellate cells (shown in blue) are laden with perinuclear retinoid droplets and preservethe differentiated function of surrounding cells, including hepatocytes and sinusoidal endothelial cells. In liverinjury, the cells multiply, lose vitamin A and become embedded within dense extracellular matrix. This leadsto deterioration of hepatocyte function manifested as loss of microvilli, and decreased size and number ofendothelial fenestrations. Reprinted, with permission, from (68).

by highlighting the accelerating pace of progress and increas-ingly nuanced understanding of a cell type that is unique notonly in liver, but throughout mammalian biology. The fas-cination with stellate cells may explain why the publishedliterature contains more than 2800 articles about this cell typeonly in the last ten years (Pubmed search using the keyword“hepatic stellate cell” from 2002-2012).

Hepatic Stellate Cell Biology, Originand UltrastructureHSCs are resident nonparenchymal cells located in the suben-dothelial space of Disse, between the basolateral surface ofhepatocytes and the antiluminal side of sinusoidal endothe-lial cells (Fig. 1). This privileged location, together with theirdendritic cytoplasmic processes, facilitates their direct con-tact with hepatocytes, endothelial cells, other stellate cells,and Kupffer cells up to 140 μm away (86, 126). This intimatecontact between stellate cells and their neighboring cell typesmay facilitate intercellular transport of soluble mediators andcytokines. In addition, stellate cells are directly adjacent tonerve endings (19, 299), which is consistent with reportsidentifying neurotrophin receptors (36, 137, 284, 333), andwith functional studies confirming neurohumoral responsive-ness of stellate cells (158, 203, 249). Interestingly, apart fromthe different patterns of distribution (pericentral vs. periportalpredominances) among species (28, 65, 305), stellate cellsonly represent ∼10% of the total number of resident cells innormal liver (86) and ∼1.5% of total liver volume, a low pro-portion of the total cell number in contrast to their remarkablydivergent functions in normal and diseased liver.

Prominent dendritic cytoplasmic processes from stellatecells contact hepatocytes and endothelial cells (65, 266,302, 304). These subendothelial processes have three cellsurfaces: inner, outer, and lateral. The inner one is smooth andadheres to the adluminal (basal) surface of the liver sinusoidal

endothelial cells (LSECs) while the outer surface, facingthe space of Disse, has numerous micro-projections whichcontact with hepatocytes and may function in detectingchemotactic signals, and transmitting them to the cell’smechanical apparatus to generate a contractile force thatregulates blood flow (180). Actin filaments and microtubulesare distributed in both the periphery and the core of the cell’sprocesses, respectively, and could be responsible for theirextension and retraction (115, 148, 204, 246, 252). While noelectron dense basement membrane can be identified withinthe perisinusoidal space of Disse, basement membraneproteins, including type IV collagen, nidogen, and laminin,have been identified, which are thought be functionallyimportant in preserving differentiated hepatocellular functionand stellate cell quiescence (20, 29, 70).

HSCs were first described by Kupffer in 1876 (303), usinga gold chloride method for detection of neuronal componentsin the liver. Their star-shaped characteristics led Kupffer tocall them “sternzellen” (“star cell,” in German). However, in1898, a misinterpretation based on India ink staining led himto conclude that they were actually “special endothelial cellsof the sinusoids with phagocytic capacity,” thereby confusingthem with macrophages (now often called “Kupffer cells”(304)). Ironically, recent studies have indeed established thatstellate cells have phagocytic properties (35, 129, 190, 301).

Since Kupffer first identified stellate cells nearly 150years ago (303), a number of new techniques for their detec-tion and isolation have been developed based on their lipiddroplet content, cytoskeletal features, and cell surface mark-ers (288). These approaches have aided in further definingtheir ultrastructure and enhancing their purity during isola-tion, for example, by using vitamin A fluorescence to isolatethe cells using flow cytometry (45). In normal liver, stel-late cells have spindle-shaped cell bodies with perikaryonswithin recesses between neighboring parenchymal cells, withoval or elongated nuclei. Vitamin A lipid droplets are con-spicuous features of the cytoplasm. Stellate cells also have

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Comprehensive Physiology Hepatic Stellate Cells and Liver Fibrosis

well-developed juxtanuclear small Golgi complex and roughendoplasmic reticulum spaces indicating active biosynthe-sis of secreted polypeptides or proteins (56). The presenceof active lysosomes in stellate cell cytoplasm described fordecades (33, 129) is now recognized to indicate a high capac-ity for the cells to undergo autophagy during cellular acti-vation (104, 105). Endosomes and multivesicular bodies arealso present in HSCs (267) and contribute to the generationof vitamin A-containing lipid droplets.

In contrast to the well-established origins of other liverpopulations (endoderm-derived hepatocytes and cholangio-cytes, mesoderm-derived endothelial cells and fibroblasts,and ectoderm-derived neurons), the ontogeny of this enig-matic cell has been controversial for decades, because thecells express a pattern of cytoskeletal markers reflecting arange of origins including ectoderm (e.g., glial fibrillary acidicprotein (GFAP), nestin, neurotrophins and their receptors,nerve growth factor (NGF), brain-derived neurotrophic factor,synaptophysin, and N-CAM) and mesoderm (e.g., vimentin,desmin, alpha smooth muscle actin, hematopoietic markers)(80, 186).

More recently, elegant developmental studies have estab-lished that stellate cells are traced to mesothelial cells(likely derived from septum transversum mesenchyme),which appear to give rise to cells that invade the hepaticbud (8, 11, 165). However, due to the limited labeling effi-ciency of the mesothelium, the proportion of stellate cellsderived from the mesothelium is not known and other cel-lular sources may also be possible. There is ample evidencethat HSCs are remarkably heterogeneous in their content ofretinoid, cytoskeletal phenotype, potential for activation, andeven their capacity to revert to a quiescent state after liverinjury resolves (45, 80, 143, 170). For example, there is asubpopulation of stellate cells that may lack typical cytoskele-tal markers (16, 235, 257) depending on the lobular location.Pericentral areas are rich in stellate cells with longer cytoplas-mic processes (i.e., more astrocytic morphology), predomi-nant GFAP expression (instead of desmin), decreased numberand size of lipid droplets, and more differentiated. Peripor-tal stellate cells are typically desmin positive with shortercytoplasmic processes (with a more contractile phenotype),contain more and larger lipid droplets and may be less differ-entiated (82, 195, 304, 305).

While the origin of stellate cells is becoming less con-troversial, still uncertain is the possibility that stellate cellsare pluripotent and can give rise to multiple cell lineages.This phenomenon has been reported in at least two studies(154, 324), but based only on cell culture findings, wheremodulation of cellular phenotype is notoriously promiscuousand may not reflect events in vivo. On the other hand, it isincreasingly clear that HSCs are intimately associated withthe progenitor cell niche and typically surround cells thathave stem cell-like properties (39, 40, 89, 219, 311). Thesefindings indicate a strong likelihood that stellate cells supportstem cell expansion, although underlying mechanisms andmediators that drive this interaction are not known.

Extrahepatic stellate cells have also been describedthroughout many organs in particular pancreas, where theyretain their characteristic shape and markers (267). Stellatecells in other tissues typically store vitamin A and synthe-size and secrete ECM components. Best characterized amongthese are pancreatic stellate cells, which clearly contributeto pancreatic fibrosis and tumor stroma (4, 5, 57). Pancre-atic stellate cells reportedly generate stem cells that may alsodirectly contribute to liver regeneration through differentia-tion into hepatocytes and duct-forming cholangiocytes acrosstissue boundaries, but this observation requires confirmation(153).

Role of Hepatic Stellate Cells inNormal LiverBecause of their recognized role in hepatic fibrosis, moststudies of HSCs have focused primarily on their behaviorduring liver injury, but have neglected their contribution tonormal liver homeostasis. With an increased availability oftools to selectively express transgenes in stellate cells, theirrole in normal liver development and function is now beingilluminated (Fig. 2).

Contribution of stellate cells toliver developmentAs noted above, stellate cells can be identified within theprogenitor cell niche (near the canals of Hering) in normal,developing, and regenerating liver (248, 326). Additionally,murine fetal liver-derived Thy1+ cells, which express classi-cal markers of HSCs (α-SMA, desmin, and vimentin), pro-mote maturation of hepatic progenitors through cell-cell con-tact in culture (12). Pleiotrophin, a morphogen secreted bystellate cell precursors (ALCAM+ submesothelial cells) dur-ing liver development, may contribute to liver organogenesisand regeneration (9, 10). Quiescent stellate cells also expressepimorphin, a mesenchymal morphogenic protein involved

Drug metabolismand detoxification

Retinoid metabolism

Normal liverdevelopment

Quiescentstellate cell

Extracellular matrixhomeostasis

Vasoregulation

Preservation ofhepatocyte mass

Figure 2 Roles of hepatic stellate cells in normal liver.



in differentiation of rat hepatic stem-like cells by a puta-tive epithelial-mesenchymal contact that promotes bile ductepithelial morphogenesis (184), which involves the RhoA andC/EBPβ pathways. These findings complement evidence ofparacrine interactions between bile duct epithelium and eitherstellate cells or portal fibroblasts both in culture (156, 166)and in vivo (141, 156, 285). While not limited to stellate cell-bile duct crosstalk, components of the Notch (254) and Wntpathways (178, 330), purinergic signaling (52), chemokines(156) and the Dlk1 protein (298, 330) are also important inhepatic development. Stellate cell precursors, isolated fromfetal liver based on UV fluorescence in flow cytometry (157),display extensive proliferative activity, and a high capacityto express hepatocyte growth factor (HGF), CXCL12, andhomeobox transcription factors, supporting their potentialcontributions to both hepatic development and hematopoiesis(157). A recent study has identified stellate cells in zebrafishusing a reporter gene driven by the Hand2 promoter (325).Use of this model will further clarify the stellate cell’s contri-butions to hepatic development and homeostasis.

Retinoid metabolism (see section onPerpetuation, Section V, below)Vitamin A (retinoid) is primarily stored in the liver inmammals, and among liver cell types stellate cells are theprimary cellular depot (303). Normally, dietary retinoids areabsorbed by the gut and transported in chylomicron remnantsas retinyl esters to hepatocytes, where they are hydrolyzedinto free retinol. Retinol is then transferred to stellatecells, where they are reesterified (101). Importantly, thesedroplets contain not only retinoids, but also triglycerides,phospholipids, cholesterol, and free fatty acids, among others(188, 320). Recent studies have identified a family of proteinsthat coat lipid droplets known as perilipins (27,28, 253). Oneperilipin, adipose-differentiation-related protein, is expressedby stellate cells and its levels are reduced as the cells activateand lose retinoid droplets (161). The contributions of theselipid droplets go beyond the simple storage of Vitamin A, andextend to the regulation of stellate cell activation (206, 207),possibly through the impact of lipids in fueling autophagy(103, 104). The importance of these retinoid mechanismsin fibrosis however has been challenged, however, as micedeficient in lecithin retinol acetyl transferase (LRAT), theenzyme that catalyzes the esterification of retinol into retinylesters nonetheless undergo fibrosis (144) following toxicliver injury, perhaps indicating an alternate or more complexrole for retinoid metabolism in hepatic and stellate cellhomeostasis.

Extracellular matrix homeostasisIn normal liver, the ECM comprises ∼0.5% of the total liverweight (245) and is distributed between portal triads, centralveins and Glisson’s capsule, with only a small portion presentin the space of Disse (81). Normal ECM components include

collagens (type I, III, IV, V, VI, XIV, and XVIII), elastin,structural glycoproteins (laminin, fibronectin, nidogen/entactin, tenascin, osteopontin, and secreted acidic proteinsrich in cysteine), proteoglycans (heparan sulfate, chondroitin4-sulfate, chondroitin 6-sulfate, and dermatan sulfate, synde-can, biglycan, and decorin), and the free glycosaminoglycanhyaluronan (74, 136, 259). While quantitatively modest in thelatter location, ECM in the space of Disse has an importantrole in preserving liver homeostasis and has a unique spa-tial expression pattern. Type IV collagen is primarily locatedbetween LSECs and stellate cells while type I and III fibrillarcollagens are located between HCSs and hepatocytes in nor-mal liver (20, 70). Primarily, the ECM composition can affectthe behavior of surrounding liver cells through cell surfacereceptors, especially integrins, of which stellate cells expressα1β1, α1β2 (227), αvβ6 (222), α5 β1 (110), αvβ6 (216,222), as well as integrin linked kinase (269).

The three cell types surrounding the space of Disse (hep-atocytes, endothelial cells, and stellate cells) each produceECM components in normal liver. While all of them expresscollagen type I, hepatocytes mainly produce fibronectin (233),endothelial cells express collagen IV, and quiescent stellatecells secrete laminin and collagen types III and IV (83, 171),among several other ECM proteins.

The maintenance of ECM homeostasis requires turnoverin which production of new components is offset by parallelrates of degradation. Matrix metalloproteinases (MMPs) arethe primary effectors of ECM degradation, whose activity isregulated in turn by tissue inhibitors of metalloproteinases(TIMPs) (6, 145). Several liver cell populations (i.e., Kupf-fer cells, myofibroblast, and hepatocytes) can produce bothMMPs and TIMPs (6, 145), however a subgroup of this fam-ily, the A Disintegrin and Metalloproteinase-domain proteins(ADAMs) may elude TIMP action and contribute to trans-forming growth factor beta (TGF-β) activation (26), the mostpotent stimulus for collagen I production by stellate cells (91,116, 296). While most ADAMs are expressed by more thanone liver cell type, at least two (ADAM 13 and 28) are pro-duced solely by stellate cells (194, 261, 273, 314).

Secretion of mediatorsWhile stellate cell-derived molecules are a major drivingforce in hepatic fibrosis they may also play an importantrole in preserving liver homeostasis and promoting regener-ation, although the data do not fully support such a role yet.The specific spatial and temporal expression patterns of thesemolecules may therefore be important to promoting properhepatic development and regeneration after injury. In steadystate conditions, stellate cells are reported to secrete a rangeof molecules detailed in the following sections.

Growth factors

HGF is the most potent mitogen for hepatocytes (256). Quies-cent stellate cells can produce HGF, but interestingly, during



liver damage the expression of this growth factor is downreg-ulated in stellate cells by the action of transforming growthfactor β (TGF-β) (232). This temporal expression of HGFmay, therefore, explain the decreased rate of hepatic regener-ation in a fibrotic/injured liver.

TGF-β is among most potent cytokines that regulate stel-late cell phenotype (21, 51). In normal liver TGF-β iso-form expression (TGF-β1,2,3) is shared between hepatocytes,Kupffer cells and stellate cells (21). While TGF-β1 is morehighly expressed by Kupffer cells than HSCs, TGF-β3 is onlyexpressed by stellate cells (48). Regardless, TGF-β is secretedin its latent form, and requires a further activation of the latentmolecule to exert its action. The predominant pathways ofTGF-β activation diverge among different tissues. In liver,integrins, fibrinogen, and urokinase-type plasminogen acti-vator, among others, can activate latent TGF-β during liverinjury, which eventually induces stellate cell activation (25,227, 278). On the other hand, it can be inactivated by bindingto the proteoglycan decorin (15). The recent elucidation ofthe latent TGF-β structure (271) could yield important newapproaches to selectively blocking its activation in vivo.

Vascular endothelial growth factor (VEGF) is alsoexpressed by quiescent stellate cells (316). Its potent mito-genic effect toward sinusoidal and endothelial cells under-scores the key role that stellate cells play in communicationand control of liver homeostasis. This important paracrine sig-naling pathway between stellate cells and sinusoidal endothe-lium, mediated by VEGF and soluble guanylate cyclase, maybe critical for sinusoidal homeostasis in normal liver andregeneration (309, 310, 319).

Other growth factors synthesized by stellate cells areinsulin-like growth factors (IGF-I and IGF-II), transforminggrowth factor α (TGF-α), EGF, stem cell factor, and fibrob-last growth factors (both acidic and basic FGF), although theircontributions may be more critical during liver developmentand regeneration (39, 40, 75, 177, 182, 191, 247, 298, 332).

Neurotrophins and their receptors

NGF, brain-derived neurotrophin, neurotrophin 3, neu-rotrophin 4/5, the low-affinity NGF receptor p75 and the high-affinity tyrosine kinase receptors B and C are all expressedby HSCs (36), and/or their precursors (284). A number ofpotential functions of these pathways are suggested by theiractivities in other tissues; however, to date, their best knownfunction in liver is in contributing to stellate cell activationand tissue repair (36, 137, 215).

Other mediators

Endothelin-1 (ET-1) is a potent vasoconstrictor produced pri-marily by endothelial cells in normal liver (322), but alsoby stellate cells. Interestingly, during liver injury, endothe-lial cells decrease their production and stellate cells becomethe dominant source of ET-1, which correlates with stel-late cell activation (138, 221, 242, 270) highlighting thecomplex interplay of ET-1 between stellate and endothelial

cells. A carefully regulated pathway exists for activation oflatent endothelin in stellate cells (138, 164).

Human stellate cells have also functional receptors foradrenomedullin (ADM), a peptide produced by most contrac-tile cells, which modulates the contractile effect of ET-1 (88).Moreover, cultured human stellate cells secrete ADM in base-line conditions, and its production is markedly increased bycytokines (88), a surprising finding given that stellate cell acti-vation promotes cellular contraction. ADM can also attenuateactivation of stellate cells by inhibiting TGF-β1 productionand TGF-β-induced MMP-2 expression partially through theERK pathway (312). These results suggest that ADM regu-lates stellate cell activation and contractility in an autocrinemanner.

A more comprehensive assessment of protein productionby stellate cells was generated by an unbiased proteomic anal-ysis of the cells and their surrounding ECM (13, 127, 236).In initial work by Kristensen, the patterns of protein expres-sion were compared between quiescent, in vivo activated andin vitro activated stellate cells, by two-dimensional-gel elec-trophoresis. From the 300 identified proteins, 83 were found tobe secreted, including collagen α1 (I), α1 (III), and α2 (I); α1-antitrypsin; calcyclin, calgizzarin, and galectin-1; proteasesincluding plasminogen activator inhibitor-1 and cathepsin A,B, and D; ganglioside GM2; among others. A more recentanalysis by Ji et al. has emphasized the importance of stellatecells in also generating immunoregulatory molecules, con-sistent with their function in conferring immune tolerance inliver (130, 290, 318).

Drug metabolism and detoxificationHSCs express both alcohol- and acetaldehyde-dehydrogenases, but not cytochrome P450-2E1 (34)and it is likely that their contribution to ethanol detoxificationis minimal compared to hepatocytes. Apart from P450-2E1,other isoforms of cytochrome p450 are expressed by stellatecells, and are downregulated during cellular activation (321);however, their roles in cellular quiescence and activation areunknown. Some cytochrome p450 isoforms have been iden-tified in stellate cells (160), implicating their participation inxenobiotic detoxification and oxidant stress response.

Role of Hepatic Stellate Cells in LiverInjury and FibrosisThe framework for understanding stellate cell activation wasestablished several years ago (63), and remains a practicaland relevant template for characterizing the cell’s response toinjury. A common consequence of liver injury is parenchy-mal damage with an increase in apoptotic bodies, Kupffercell activation, production of oxidative species, and ECMremodeling (65), which all function as triggers for cellu-lar activation. Activation of stellate cells comprises twowell-established phases: initiation (also called “preinflamma-tory stage”) and perpetuation, which can be followed by a



potential third phase, resolution, if the liver injury resolves(58, 64, 69). During resolution of fibrosis, loss of activatedstellate cells occurs through numerous pathways and there isnow evidence that stellate cells can not only undergo apopto-sis, but are also able to either become senescent or revert to aquiescent phenotype (67, 143, 294). The elucidation of molec-ular mechanisms underlying these events may accelerate thediscovery of potential antifibrotic drug targets.

Initiation of stellate cell activationStellate cell initiation promotes changes in gene expressionand phenotype that render the cells susceptible to the changingenvironment and stimuli in the injured liver, thereby promot-ing the transition to the perpetuation phase. The earliest sig-nals triggering the initiation of stellate cell activation resultfrom paracrine stimulation by neighboring cell populations(endothelial cells, platelets, immune cells, and hepatocytes)and changes in its surrounding ECM (Fig. 3).

As endothelial cells comprise the vascular lining of theliver’s sinusoids, they play a vital role in these early stages.They induce the activation of stellate cells by secretingfibronectin (125) and by activating latent transforming TGF-β(149), as well as through secretion of a range of mediators thatmodulate inflammation and participate in cellular crosstalk(275, 319). Fibronectin’s effects are largely promigratory andnot fibrogenic, suggesting that stellate cell migration is animportant first step in responding to injury (210). Platelets alsocontribute by secreting TGF-β, as well as EGF and platelet-derived growth factor (PDGF), the most potent stellate cellmitogen identified (14, 24). Overall however, the residenthepatic macrophage population may be the main source ofPDGF, as well as other paracrine mediators that drive stellatecell activation (106, 287, 308).

There is an increasingly nuanced understanding ofhow inflammatory and immune cells regulate stellate cellresponses and activation. In particular, T cells, dendritic cells(DC), and macrophage subsets all have well defined inter-actions with stellate cells [see (76, 131, 152, 179, 313) forreviews]. Among these, recent studies have characterized aspecific macrophage subset in rodents, Ly-6Clo, that are vitalfor regression of hepatic fibrosis (231). On the other hand,different macrophages can drive stellate cell function includ-ing stimulation of matrix synthesis, cell proliferation, andretinoid release by secreting TGF-β, TNF-α, and MMP-9, andproduction of reactive oxygen species (ROS) and lipid perox-ides (230). Moreover, ROS produced by hepatic macrophagescan initiate downstream signals that include osteopontin, anECM protein that can induce collagen (300) and perpetuatethe activated stellate cell phenotype. Recent studies furtherimplicate an inhibitory role of platelets in blocking stellatecell activation based on the following: (i) transgenic throm-bocytopenic mice develop exacerbated liver fibrosis, withincreased expression of type I collagen α1 and α2, duringcholestasis (147); (ii) in vitro experiments reveal that, uponexposure to stellate cells, platelets became activated, released

HGF, and then inhibited stellate cell expression of the type Icollagen gene in a Met signal-dependent manner (147); and,(iii) activation of human stellate cells in culture is suppressedby human platelets or platelet-derived ATP via the adenosine-cAMP signaling pathway (114).

Hepatocytes are the main target for most forms of liverinjury including viral infection, alcohol, and obesity, amongothers (198). Following injury, damaged hepatocytes becomea major source of lipid peroxides and apoptotic bodiesthat initiate stellate activation through a process mediatedby Fas and TRAIL (32). The contribution of hepatocyte-derived apoptotic bodies to stellate cell activation is indepen-dent of the inflammatory response, since in cultured stellatecells, addition of hepatocyte apoptotic fragments are directlyfibrogenic (33), and can also activate Kupffer cells (31).Hepatocytes also express P450-2E1, an important enzymeinvolved in the metabolism of xenobiotics as ethanol, and apotent source of ROS (193) that can stimulate stellate cellfibrogenesis (193).

Changes in the composition and stiffness of ECM alsoimpact on stellate cell responses (84, 121, 315) suggestinga feed-forward loop where stellate cell mediated changes inECM further drive stellate cell activation. Early changes intranscription factor activity in response to ECM, as well assoluble signals set the stage for a broad phenotypic transi-tion of the cells, and a large number of nuclear factors havebeen implicated [see (173) for review]. Additionally, path-ways of translational, transcriptional and posttranscriptionalregulatory control (including epigenetic pathways and miR-NAs) contribute to this process (18, 41, 99, 163, 172, 174,218, 243, 297).

While the initial presumption that transcription factorslargely stimulate stellate cell activation, it appears equallytrue that other factors repress activation, and their activity isdownregulated during cellular activation. Three key examplesinclude Lhx2 (306), KLF6 (85), and Foxf1 (133), in whicheach contribute to preservation of a quiescent phenotype, suchthat their loss or downregulation derepresses the activationprogram.

Perpetuation of stellate cellactivation—mechanisms and implicationsAfter the initial liver injury, stellate cells initiate activationfollowed by a process of perpetuation, leading to accumula-tion of ECM and culminating in the formation of scar tissue.Perpetuation of stellate cell activation is a tightly orches-trated process that includes a number of functional responsesincluding proliferation, fibrogenesis, chemotaxis, contractil-ity, matrix degradation, retinoid loss, and cytokine/chemokineexpression (Fig. 4).

Proliferation

PDGF is most potent stellate cell mitogen during liverinjury. An increase in available PDGF and stellate cellresponsiveness due to increased expression of PDGF receptor


Joseph George

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Fibril-forming collagens (types I, III, and V)Basement membrane collagens (type IV and VI)Glycoconjugates (laminin, fibronectin, glycosaminoglycans, and tensacin)

Hernandez-Gea V, Friedman SL. 2011.

Annu. Rev. Pathol. Mech. Dis. 6:425–56

Increase in fibril-forming

collagen in space of Disse

Distortion

of veinsLoss of endothelial fenestrations

(B)

HSC

Terminal

hepatic

vein

Portal vein KCSinusoidal endothelial cells

Sinusoidal space of Disse

Normal liver

Fibrotic liver

(A)

Portal triad

Hepatic

arteriole

Hepatocytes

Loss of hepatocyte

microvilli

HSC activation

and proliferation

Bile duct

Figure 3 Matrix and cellular alteration in hepatic fibrosis. Normal liver parenchyma contains epithelial cells (hepatocytes) andnonparenchymal cells: fenestrated sinusoidal endothelium, hepatic stellate cells (HSCs), and Kupffer cells (KCs). (A) Sinusoids areseparated from hepatocytes by a low-density basement membrane-like matrix confined to the space of Disse, which ensures metabolicexchange. Upon injury, the stellate cells become activated and secrete large amounts of extracellular matrix (ECM), resulting inprogressive thickening of the septa. (B) Deposition of ECM in the space of Disse leads to the loss of both endothelial fenestrations andhepatocyte microvilli, which results in both the impairment of normal bidirectional metabolic exchange between portal venous flowand hepatocytes and the development of portal hypertension.



Oxidative stressApoptotic bodies

LPSParacrine stimuli

Initiation Perpetuation

Injury

Reversion

Apoptosis

Proliferation

Resolution

Inflammatory

signaling

HSC

chemotaxis

Altered matrix

degradation

Fibrogenesis

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PDGFVEGFFGF

ET–1NO

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TIMP-1,2

PDGFChemokines

ChemokinesTLR ligands

Adenosine

TIMP-1,2TRAIL

Fas T cellsB cells

NK cellsNK-T cells

Figure 4 Pathways of hepatic stellate cell activation and loss during liver injury and resolution.Features of stellate cell activation can be distinguished between those that stimulate initiation and thosethat contribute to perpetuation. Initiation is provoked by soluble stimuli that include oxidant stress signals(reactive oxygen intermediates), apoptotic bodies, lipopolysaccharide (LPS), and paracrine stimuli fromneighboring cell types including hepatic macrophages (Kupffer cells), sinusoidal endothelium, andhepatocytes. Perpetuation follows, characterized by a number of specific phenotypic changes includingproliferation, contractility, fibrogenesis, altered matrix degradation, chemotaxis, and inflammatorysignaling. During resolution of hepatic fibrosis, there is both programmed cell death (apoptosis) toclear fibrogenic cells, as well as reversion to a more quiescient phenotype. FGF, fibroblast growthfactor; ET-1, endothelin-1; NK, natural killer; NO, nitric oxide; MT, membrane type. Reprinted, withpermission, from (66).

results in rapid proliferation and an overall increase inthe absolute number of stellate cells with a profibrogeneicphenotype (24, 220, 317). Tumor necrosis factor (TNF) alphasignaling also contributes to PDGF-mediated stellate cellproliferation primarily through the TNF receptor 1 (291).Stellate cells are also responsive to a wide array of factorsincluding, VEGF (327), thrombin, EGF, keratinocytre growthfactor (282), and bFGF (328). Among those, VEGF is one ofthe major cytokines secreted by activated HSCs, which drivesboth angiogenesis and fibrogenesis, as described above (44,135, 159). VEGF production is dependent on the overex-pression of COX-2 protein via phospho-p42/44 MAP kinaseactivation (329). Overall, HSCs contribute to both woundhealing and tumor growth, a conclusion underscored by sev-eral studies implicating this cell type in the development andgrowth of both primary and metastatic tumors (47, 134, 209).

Tissue inhibitors of matrix metalloproteinases (TIMPs)are profibrogenic by inhibiting matrix degradation, and pro-moting stellate cell survival. Increased TIMP-1 expression

by stellate cells also drives stellate cell proliferation in anAKT-dependent manner (61). This mechanism might con-tribute to the antiproliferative effects of activated VitaminD, 1,25(OH)(2)D(3) (1). Vitamin D receptor is expressed byquiescent stellate cells (79), and its expression is downregu-lated with activation. Consequently, treatment of stellate cellswith 1,25(OH)(2)D(3) dampens proliferation via cyclin D1suppression and decreases expression of type I collagen andTIMP-1 while simultaneously increasing MMP-9 expression.Patients with liver disease exhibit vitamin D deficiency, andappropriate supplementation might prove to be a useful ther-apeutic intervention (225).

MicroRNAs are small 19-24 noncoding RNA sequencesthat can regulate posttransciptional gene expression bysequence-specific binding to the 3′-UTR of mRNAs to pro-mote their degradation. The role of miRNAs in fibrosis pro-gression is being clarified, as miRNA levels change in liversof patients with fibrotic disease and in stellate cells duringactivation (93, 99, 112, 196, 197, 205, 243, 307). In stellate



cells, miRNAs control proliferation and fibrogenesisby regulating protein expression of proproliferative andprofibrogenic signaling pathways. In particular, miR-27a and-27b and miR29b are upregulated in activated stellate cells,whereas their suppression leads to decreased proliferation andan increase in lipid droplets indicative of the quiescent pheno-type (205, 244). miR-27a -27b directly target the 3′-UTR ofretinoid X receptor α (RXRα) to inhibit its expression. RXRα

can decrease DNA synthesis, leading to growth arrest in stel-late cells (100). Furthermore, RXRα regulates adipogenesisby activation of peroxisome proliferator-activated receptor γ

(PPARγ) which is a master regulator of stellate cell activation(297). RXRα expression is decreased in stellate cell activa-tion and its expression in activated stellate cells increases withinactivation of miR-27a -27b, indicating a direct interactionbetween RXRα and miR27a -27b (128). In contrast, miR-195 is downregulated during stellate cell proliferation andits expression is induced upon treatment with IFN-β, whichexhibits antifibrotic effects independent of its antiviral activ-ity. Treatment with IFN-β downregulates cyclin E1 and upreg-ulates p21 in a miR-195 specific manner thereby promotingcell cycle arrest and decreased stellate cell proliferation (265).

Fibrogenesis

Production of ECM, in particular collagen type I, is a hallmarkof activated stellate cells. Production of collagen type I bystellate cells is regulated both transcriptionally and posttran-sciptionally (2, 37, 73, 117-119, 167, 175, 214, 238, 279,280,295). TGF-β1 is major driver of this process through autocrineand paracrine stimulation of ECM production (see above).The other well-characterized fibrogenic cytokine towards stel-late cells is connective tissue growth factor (CTGF/CCN2).Levels of CTGF are increased in liver injury and the cytokinepromotes a range of profibrotic activities toward stellate cells,mediated by a G-coupled protein receptor (78, 92, 111). CTGFrepresents a very appealing target for antifibrotic therapy,since unlike antagonism of TGF-β1, CTGF inhibition shouldhave no impact on hepatocyte growth or confer a risk of car-cinogenesis.

There is a growing list of other factors that contributeto fibrogenesis, including signaling molecules, chemokines,and cellular stressors (250). For example, osteopontin, anECM cytokine expressed by stellate cells, activates collagenI expression via integrin α(V)β(3) engagement and activa-tion of the PI3K/pAkT/NFκB signaling pathways (300). Fur-thermore, the recent identification of receptors to profibro-genic chemokines on stellate cells including CXCR4 (108),CCR1, CCR5 (262), CXCR2 (281), and CCR2 (263), addto the repertoire of signals promoting stellate cell activation.The targetability of chemokine receptors with small moleculeinhibitors makes them ideal candidates for antifibrotic ther-apies. Recently, blockade of IL-17A has been proposed asa potential strategy for cirrhosis treatment due to its induc-tion (together with its receptor) in response to liver injury.IL-17A may promote fibrosis by activating inflammatory and

liver resident cells and inducing collagen type I productionin HSCs through engagement of the signal transducer andactivator of transcription 3 signaling pathway (181).

As discussed above, (see “Proliferation”) miRNAs playa significant role in stellate cell biology and can modulatecollagen synthesis. MiR-29b binds directly to the 3′-UTR ofcollagen-IαI and -IV, thereby inhibiting its translation. miR-29b expression is repressed by TGF-β, and its overexpressioninhibits TGF-β induced collagen expression via a SMAD-independent mechanism, while HGF, which exhibits antifi-brotic effects in stellate cells, induces miR-29b expression(244, 266). MiR-21, whose expression is enhanced duringfibrotic disease, is also controlled by TGF-β signaling. TGF-β functions via 2 distinct mechanisms to increase miR-21production. Smad3 induces miR-21 transcription, while bothSmad2 and Smad3 enhance miR-21 maturation.

Chemotaxis

Stellate cell chemotaxis is an important event in the gener-ation of fibrotic septae by allowing activated cells to alignwithin regions of injury. Stellate cells migrate primarilytowards chemoattractant cytokines (chemokines), and theyexpress a range of chemokine receptors and their cognatechemokine ligands. Notably, stellate cells migrate towardsPDGF (113, 142), VEGF, Ang-1 (199), TGF-β1, EGF (323),b-FGF (59), CCL2 (176), and CXCR4 (254), and CXCR3specific ligands (23). The mechanism of chemotaxis includesa cytoskeletal remodeling with cell spreading at the tip, move-ment of the cell body towards the stimulus, and retraction oftrailing protrusions (180). Oxidant signaling contributes tothese responses. Specifically, numerous chemoattractant sig-nals (PDGF, VEGF, and CCL2) increase NADPH oxidase-dependent intracellular ROS and activation of the ERK1/2and JNK1/2 pathways (50, 212). Furthermore, generation ofintracellular superoxide anion or H2O2 by treatment withmenadione promotes cell migration even in the absence ofspecific chemoattractants (198).

Hypoxia is another broad activator of stellate cell migra-tion, which functions via two distinct mechanisms. Afterinduction of hypoxic conditions, mitochondrial-generatedROS activate the ERK1/2 and JNK1/2 pathways, drivingmigration. Sustained hypoxia leads to a HIF-1α-dependentincreased production and secretion of VEGF by stellate cells,promoting their mobility (200).

Since stellate cell mobilization is also required for tis-sue wound healing, it has been reported that the space ofDisse microenvironment, per se, is another key factor in reg-ulating the migratory behavior of stellate cells. ECM com-ponents including MMP-2 and type I collagen are able toinduce stellate cell migration (208, 323). Cellular fibronectincontaining an alternatively spliced domain A (EIIA) is upreg-ulated during liver injury. Signaling specifically by the EIIAfibronectin variant though integrin receptor α(9)β(1) on stel-late cells promotes formation of lamellipodia and cellularmotility, further implicating ECM signaling in stellate cell



biology (210). The hyaluronic acid receptor (CD44) is alsoincreased in liver injury and repair, promoting both activationand migration of stellate cells (140). Interestingly, a specificsplice variant (CD44v6) is responsible for up to 50% of thismigration, confirming the idea that activated stellate cells maydepend, to some degree, on CD44v6 and hyaluronic acid formigration.

Contraction

Stellate cell contraction is thought to be a primary determi-nant of portal hypertension in patients with end-stage liverdisease. The factors leading to portal hypertension includeincreased blood flow, increased intrahepatic resistance, anddisrupted liver architecture. During injury, the hepatic sinu-soids undergo both morphological and functional changesmediated by HSCs. Dramatic remodeling occurs, character-ized by deposition of collagen matrix, loss of fenestrationsand increased density of contractile HSCs (293). Addition-ally, there is an imbalance of vasoactive forces characterizedby deficient nitric oxide production and an increase in vaso-constrictive substances including ET-1, angiotensinogen II,eicosanoids, atrial natriuretic peptide, somatostatin, and car-bon monoxide (237, 239, 240, 292). Together, these factorslead to an increase in sinusoidal resistance and portal hyper-tenstion.

The final step in the induction of cellular contractionis phosphorylation of MLC-2, resulting from a calcium-dependent and a calcium-sensitive pathway. In the calcium-dependent pathway, typically in skeletal and cardiac muscle,release of Ca2+ from the endoplasmic/sarcoplasmic reticulumleads to activation of myosin light-chain kinase (MLCK) bycalmodulin and subsequent phosphorylation of MLC. Alter-natively, in the Ca2+-sensitive pathway, the dominant path-way in stellate cell mediated contraction and smooth mus-cle cells, Rho-kinase inactivates the myosin binding subunitby phosphorylation, thereby preventing it from dephospho-rylating MLC and inhibiting contraction. The net effect ofRho-kinase activation is increased phosphorylated-MLC andcontraction (168).

Adenosine plays an important role in stellate cell dif-ferentiation, proliferation, and type I collagen production.Despite these profibrotic effects, however, adenosine inhibitsstellate cell contraction (as well as chemotaxis) via loss ofactin stress fibers. Engagement of the A2a receptor by adeno-sine promotes PKA activity and Rho A inhibition (276),which establishes a rationale for Rho antagonism as a strat-egy for treatment of portal hypertension (132). Adenosinetherefore promotes both injurious and protective effects onstellate cells and understanding these different functions willbe important in the development of antifibrotic agents. Inter-estingly, the antifibrotic activities of caffeine, now validatedin several large cohorts (43, 187, 211), may reflect the com-pound’s effect in reducing adenosine signaling in stellatecells (38).

Retinoid loss

Recently, there has been a renewed focus on the roleof retinoid loss in stellate cell activation and collagenproduction. Stellate cell activation is characterized by the lossof perinuclear retinoid droplets (65, 70); however, their func-tion in activation and fibrogenesis is only now being revealed.

Stellate cells are the largest reserve of retinoids in the body(∼60%) and conversion of retinol into retinyl ester is a hall-mark of stellate cell activation. The abundance of vitamin Ain stellate cells is heterogenous depending on the intralobularposition of the cell (80) and may be indicative of alternate acti-vation states. Quiescent stellate cells that are isolated basedon their collagen 1 expression display an increase in CYP251retinoid catabolizing cytochrome, a decrease in retinyl esters,and a more activated phenotype compared to cells isolatedbased on their buoyancy in gradient centrifugation (45).

LRAT, which catalyzes the esterification of retinol intoretinyl ester, is the sole acyltransferase found in the liver. Withstellate cell activation, LRAT expression is lost. Addition-ally, treatment with IL-1 promotes decreased LRAT expres-sion (139). Despite its apparent role in stellate cell activation,mice deficient in LRAT neither display spontaneous fibrogen-esis, nor do they exhibit increased fibrogenesis in liver injurymodels, indicating that perhaps retinoid loss is a marker ofactivation, but is not crucial for stellate cell activation (144).However, treatment with retinoic acid can decrease stellatecell activation as reflected in reduced collagen I, MMP-9, andα-SMA by inhibiting expression of TGF-β (98).

In contrast to the lack of dependence on LRAT for fibro-genesis by stellate cells, mice deficient in LRAT are protectedfrom chemical hepatocarcinogenesis. An increase in activeretinoids due to their lack of conversion to retinol storageform leads to an overall antiproliferative effect, increased p21levels, and inhibition of tumor progression (144, 272).

An additional link between retinoid metabolism to stel-late cell activation has emerged through the recognition thatthis process requires cellular autophagy (104). Specifically,hydrolysis of retinyl esters liberates fatty acids that are metab-olized by β-oxidation, generating the substrates that are essen-tial for fueling the energy-intensive pathways of cellular acti-vation. The free retinol can be detected in the extracellularmilieu under these conditions (72), but pathways enablingits cellular egress are not known. Similar to stellate cells,autophagy contributes to the intracellular catabolism of lipidsin hepatocytes, fibroblasts (274), and neurons (150, 151).Moreover, inhibition of autophagy in hepatocytes leads toreduced rates of β-oxidation and marked lipid accumulationin cytosolic lipid droplets (274).

Matrix degradation

Fibrosis is a dynamic process of matrix production and degra-dation. Fibrotic progression is characterized by the replace-ment of normal basement membrane, collagen type IV, withscar forming collagen type I. Early matrix degradation is animportant step in fibrosis and may be essential for stellate cell



migration to sites of injury. Stellate cells are the prominentsource for MMP-2, MMP-9, and MMP-13 (7, 96, 183, 302),which function as either interstitial collagenases (MMP-13)or gelatinases (MMP-2 and -9).

Despite the dependence of fibrosis progression on MMPs,these molecules paradoxically exhibit antifibrotic properties,and their expression is suppressed with fibrotic progression.MMP-2 inhibits collagen type I production by stellate cellsand mice lacking MMP-2 therefore exhibit increased fibro-sis (229). Furthermore, MMP-2 is able to regulate stellatecell apoptosis by cleavage of the extracellular domain of N-cadherin, further supporting its antifibrotic role (97). Some ofthe paradoxical activity of MMPs in vivo may be explained bytheir simultaneous activation of macrophages (87) a featureoften overlooked in interpreting their contribution to fibrosis.Regulation of MMP expression occurs through several mech-anisms. TIMPs, which are prominently expressed by stellatecells, bind MMPs, inactivating them. Additionally, with pro-gressive fibrosis, MMP-9 and MMP-13 are repressed at thechromatin level and access by the transcription factors NF-κBand AP-1 is inhibited. Impaired histone acetylation is associ-ated with permanently silenced genes, and activated stellatecells have a global increase in histone deacetlyase-4 leadingto decreases in acetylation in the MMP-9 and -13 promoterregions and gene repression (226).

While the source of enzymes that degrade ECM in liverhas been elusive, findings increasingly point to subsets ofmacrophages that have fibrolytic potential. Indeed, a recentstudy has characterized a specific subtype of macrophagesthat express the surface marker Ly-6C as a recruited cellularsubset that secretes a range of proteases that have the capacityto degrade ECM (230, 231).

Immunoregulation

In recent years stellate cells have emerged as a promi-nent determinant of hepatic immunoregulation during injury.They express of a battery of chemokines including CCL2(277), CCL5 (260), CXCL2, CCL21 (22), CXCL8, CXCL9,CXCL10, CXCL12, and CX3CL3 which have been shown torecruit neutrophils, macrophages/monocytes, NK/NKT cells,DCs, and T-cells, thereby establishing their role in immunecell infiltration. Many of these chemokines also function inde-pendent of immune cells and modulate cellular differentiation,survival, proliferation, and apoptosis.

Stellate cells can control immune cell function throughthree interrelated mechanisms (283, 288): (i) cell surfaceexpression of chemokines and/or delivery of chemokines toendothelial cells promotes lymphocyte adhesion and subse-quent migration and activated stellate cells specifically pro-mote ICAM-1 and VCAM-1 dependent adhesion and migra-tion (107); (ii) increased expression of stellate cell derivedchemokines establishes a chemoattractant gradient betweenthe peripheral blood and the liver, thus driving immune cellmigration into the liver; and (iii) stellate cell interactionwith immune cells has a direct role in promoting/inhibiting

their maturation within the liver. For example, stellate cellscan inhibit the priming of naıve T cells in a cell contact-dependent CD54 mechanism. Incubation of stellate cellswith DCs and OT-1 T-cells inhibits T cell proliferation andreduces CD25 and CD44 activation markers (255). The abil-ity of stellate cells to inhibit T cell proliferation is depen-dent on their activation state, since quiescent stellate cellsdo not possess this inhibitory function. Proteomics analy-sis reinforces this immune suppressive role (127). In thetransplantation setting, endotoxin-stimulated stellate cells areimportant immune regulators by inducing selective expan-sion of tolerance-promoting Tregs to reduce inflammationand alloimmunity (46).

Pattern recognition receptors, particularly TLR4, con-tribute significantly to stellate cell responses. Stimulationof stellate cells with the TLR4 ligands enhances TGF-βsignaling and production of inflammatory and chemotacticcytokines (CCL2, IL-6), leading to a more profibrogenicphenotype (94, 185, 213, 264). Additionally, stellate cellTLR3 activation confers antifibrotic activities by stimulatinginterleukin-10 production (30).

During late stage fibrosis when portal hypertension ispresent there is an increased bacterial load delivery from thegut to the liver due to increased intestinal permeability, leadingto an increase in LPS and TLR4 activation by stellate cells.Treatment of mice with the intestinal decontaminant rifax-imin, decreases portal pressure, fibrosis, and angiogenesis ina TLR4-specific manner (331). Specifically, rifaximin treat-ment inhibits production of stellate cell derived fibronectin.LSECs are the key mediators during angiogenesis. Stellatecell derived fibronectin is able to induce LSEC migration, andtubulogenesis, thereby contributing to the pathogenic effectsof LPS (331).

The stellate cell’s capabilities now extend to their rolein DC development (109). DCs exposed to stellate cells ortheir supernatants express low CD11c, CD86, and major his-tocompatibility complex class II, which elicits an inferiorallostimulatory function compared with conventional DC.

From the complementary point of view, the inflamma-tory response can also modulate stellate cell activation andfibrogenesis. Therefore, a positive feedback loop exists inwhich inflammatory and fibrogenic cells stimulate each otherin amplifying fibrosis. Thus, cell types regulating progressionand resolution of fibrosis include macrophages (a pivotal cellwith the potential for both a pro- and antifibrotic capacityby secreting/regulating TGF-β1, PDGF, MMPs, and TIMPs)(230,231), natural killer cells (antifibrotic activity by inhibit-ing and/or killing activated stellate cells) (77, 189), T-cells(responsible for initiating/maintaining the adaptive immuneresponse and may induce liver injury upon CCL3/MIP1α

recruitment of CCR1 expressing CD4+ T cells) (286), B-cells(fibrosis promotion in an antibody- and T cell-independentmanner) (201), DCs (involved in both proinflammatory andimmunogenic responses) (42, 131), as well as endothelial cells(antigen-presenting cells for CD4+ and CD8+ T populationswith antigen clearance activity) (146).



Stellate Cells and Resolution ofLiver FibrosisDespite the historical conception of liver fibrosis as a passiveand irreversible process due to hepatocyte collapse (223,224),the idea of fibrosis regression was proposed in the 1970s (217,224) and demonstrated in the 1990s (17, 95). At a certainstage of disease, fibrosis may become irreversible, likely dueto significant collagen cross-linking and development of aninsoluble and relatively hypocellular matrix, which may coin-cide with the appearance of clinical cirrhosis manifestations(124). Since stellate cells are an important contributor to ECMremodeling in liver fibrosis, three possibilities may accountfor regression of fibrosis, either their apoptosis, senescence,or reversion to their quiescent stage.

Apoptosis of stellate cells during liver fibrosis recoverycontributes to a decrement in the number of activated stellatecells (120, 123, 251). This situation coincides with a decreasein TIMP-1 expression (122), which protects the cells fromapoptosis and inhibits the MMPs functions (192), favoringthe partial degradation of ECM (55). Of interest, the NF-κBtranscription factor plays a role in the stellate cell’s protec-tion from apoptosis during liver fibrosis resolution, since itsinhibition can accelerate recovery from liver fibrosis (202).Moreover, apoptosis can be spontaneously initiated in acti-vated stellate cells via CD95L (Fas ligand) Bcl-2 and p53(90). Finally, other cell populations can contribute to stellatecell apoptosis. Hepatocytes can secrete NGF, which is apop-totic towards stellate cells (228) (203). Natural killer cellscan also provoke stellate cell apoptosis through TRAIL andNKG2D, a stellate cell membrane receptor that is specificallyexpressed by activated stellate cells, rendering them suscepti-ble to NK action (228). Kupffer cells can also induce stellatecell apoptosis by a caspase-9-mediated mechanism (60).

Recent studies have now established that HSCs can underop53-mediated senescence in culture (258) and in vivo (155).Initial studies in cultured HSCs suggested that as the cellsreached their replicative limit, they adopt a more inflammatoryand less fibrogenic phenotype (258). Based on these observa-tions, elegant in vivo approaches confirmed the developmentof senescence in HSCs in vivo (155, 169). Moreover, thisresponse is p53-dependent, because animals in which p53 isspecifically deleted in stellate cells have more fibrosis aftertoxic liver injury (169). Remarkably, this senescence programregulates the polarization of macrophages towards a pheno-type that is protective against experimental carcinogenesis,unearthing a noncell autonomous pathway of tumor suppres-sion by p53 through its impact on the surrounding stromalbiology (169).

Until recently, reversion from activated to quiescent HSCwas only demonstrated in cultured cells. However, elegantstudies have now used genetic methods to document reversionof activated cells to a quiescent phenotype in vivo (67, 143,294). Reverted cells, however, have a heightened capacity toreactivate, compared to those that have never activated. Quan-titatively, reversion of activated stellate cells to quiescence is

likely to be a significant pathway in fibrosis regression thatinvolves approximately 50% of the stellate cell population,and may in part be regulated by changes in PPARγ activity.

ConclusionHSCs are among the most fascinating and versatile of celltypes in mammalian biology. While their contributions to hep-atic injury and fibrosis has been well established for at leasttwo decades, clarifying this role further continues to bene-fit from remarkable discoveries uncovering the signals thatcontrol cell plasticity, survival, and intracellular signaling. Agrowing range of genes, epigenetic changes, mediators, andsecretory proteins of stellate cells has been uncovered usingcomplementary methods of gene profiling and proteomics,which has led to a more integrated understanding of the cell’sregulation. Concurrently, genetic models including new meth-ods to delete genes specifically in stellate cells further clarifytheir roles in normal and injured liver. In aggregate, these dis-coveries have enhanced the perceived importance of HSCs notonly to the fibrotic response in liver, but also to normal liverhomeostasis that includes blood flow regulation, regeneration,and stem cell responses.

A key issue in studying HSCs is the choice of modeland species. Studies over the past 25 years have utilized bothrodent and human cells, as well as several methods to immor-talize cultured cells, including SV40 T antigen gene transfec-tion, spontaneous immortalization in low serum, or ectopictelomerase gene expression. Whereas human cells have theadvantage of direct human disease relevance, they lack thecapacity for in vivo gene deletion using knockout technology,which is now widely used for mouse models and isolatedstellate cells. This raises an important issue, as yet unan-swered, whether the genotypes and phenotypes of rodent andhuman stellate cells in normal and diseased liver are suffi-ciently similar to allow investigators to rely on rodent studiesto understand and predict human disease targets. This concernhas become more pertinent based on a recent study indicatingthat rodent models of inflammation bear little resemblanceto human disease (268). While one approach might be to“humanize” mice so that they contain human stellate cells,methods to achieve this goal would be tedious and wouldlikely require use of immunodeficient animals. An alternativeapproach would be to perform comprehensive comparativeanalysis of mRNAs, microRNAs, proteins, and/or epigenet-ics between human and rodent cells from normal and injuredliver to determine how faithful rodent cells are to their humancounterparts. Based on studies to date, there is no evidencethat fibrogenic responses by stellate cells between species arewidely divergent, but the question has not yet been rigorouslyaddressed.

Despite progress in the field, several other key issues alsoremain unresolved. The contribution of stellate cells to regen-eration has long been assumed, but recent reports suggestthat HSC depletion has no effect on regeneration (54, 144).Furthermore, use of transgenic animals has begun to trace



stellate cell fate during fibrosis regression (143, 230, 294) butunderlying mechanisms are still incomplete. Similarly, it isincreasingly clear that stellate cells and the fibrotic stromathey generate in injured liver accelerate the risk of hepato-cellular carcinoma, but methods to clarify pathways linkingfibrosis to cancer are only now emerging. Also unclear is thefull range of paracrine interactions in stellate cell crosstalkwith sinusoidal endothelial cells, biliary epithelial cells, andimmune cell subsets. Finally, although activation of HSCsinto contractile myofibroblasts is well recognized as the cen-tral event in hepatic fibrosis, this insight has not yet translatedinto an effective antifibrotic therapy for patients with chronicfibrosing liver diseases. Future studies in animal models willfurther clarify these vital roles, while their translation intoeffective antifibrotic drugs for fibrotic liver disease is sure toemerge in the coming years.

AcknowledgementsWork in the authors’ laboratory was supported by NIH GrantsDK56621, AA020709 (to S.L.F.), NIDDK F30-DK090986,and T32-GM007280 (to Y.S.), and funds from the AlfonsoMartin Escudero Fundation (to J.E.P.).

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Further ReadingFriedman SL, Sheppard D, Duffield J and Violette S. Therapy for fibrotic

diseases: Nearing the starting line. Sci Transl Med, 5:167sr1, 2013.Friedman SL, guest editor. Special issue on fibrosis. Biochim Biophys Acta.

2013 Mar 16. pii: S0925-4439(13)00077-X. doi: 10.1016/j.bbadis.


hepatic stellate cells and liver fibrosis · hepatic stellate cells and liver fibrosis...

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