283 Reversibility of liver fibrosis. CONCISE , 2009; 8 (4): 283-291 REVIEW October-December, Vol. 8 No.4, 2009: 283-291 Reversibility of liver fibrosis Prakash Ramachandran,*John P. Iredale* * Centre for Inflammation Research to MRC, University of Edinburgh Centre for Inflammation Research ABSTRACT Liver cirrhosis is a major cause of morbidity and mortality worldwide and has very limited therapeutic op- tions. Regardless of the aetiology, hepatic fibrosis is a characteristic feature of chronic liver disease. Our knowledge regarding the pathogenesis of this scarring has grown exponentially in the past 25 years. It has now clear that this is a highly dynamic process and the long-held dogma that it is irreversible and relent- lessly progressive is now being challenged. In this review, we will summarise the key pathogenic mechanis- ms at play and will focus on the evidence demonstrating that liver fibrosis is reversible in humans and animal models. In particular, we will examine the role of hepatic stellate cells, MMPs, TIMPs and macro- phages in this process. Finally, we will discuss some of the studies aimed to therapeutically target the re- solution of fibrosis and their potential for translation into a badly-needed treatment modality in the clinical setting. Key words. TIMP. MMP. Macrophage. Stellate cell. Apoptosis. INTRODUCTION of complications and removal of the aetiological agent, barring liver transplantation for a select few. Liver fibrosis is the wound healing response of With an increasing incidence of cirrhosis and an al- the liver to a variety of insults. In clinical practice ready severe shortage of donor organs, it is clear no- these injurious stimuli commonly take the form of vel therapies are vital. chronic alcohol abuse, chronic viral infection with The progression from liver fibrosis to cirrhosis Hepatitis C or B, Non Alcoholic Fatty Liver Disease was long thought to be inexorable. However, recent (NAFLD) in association with obesity and the meta- research into the mechanisms of fibrogenesis has bolic syndrome, parasitic infection and a series of highlighted the dynamic nature of the process and is other autoimmune and metabolic conditions. If these beginning to challenge this dogma. In this concise insults remain chronically unchecked, ultimately review, we will focus on the evidence that hepatic fi- the scarring can progress to liver cirrhosis, with brosis is potentially reversible and highlight possi- the pathological hallmarks of advanced fibrosis, no- ble therapeutic strategies that may prove invaluable dular hepatocyte regeneration, and significant ar- in the future. chitectural disruption. Such patients become susceptible to the complications of chronic liver di- HEPATIC MYOFIBROBLASTS sease, including portal hypertension, hepatocellular ARE THE KEY SCAR PRODUCING CELLS carcinoma and liver failure. Unfortunately current IN THE FIBROTIC LIVER therapeutic options remain limited to management Following injury to the liver, there is cellular da- mage, activation of the resident inflammatory cells Correspondence and reprint request: Professor John P Iredale. as well as recruitment of additional inflammatory Professor of Medicine cells. In the acute setting the extraordinary homeos- Tissue Injury and Regeneration Group Centre for Inflammation Research tatic and regenerative capacity of the liver takes to MRD/ University of Edinburgh over, and normal architecture is restored with only 47 Little France Crescent, Edinburgh, United Kingdom, EH16 4TJ transient extracellular matrix (ECM) deposition ob- Tel.: 0131-242-6686 served.1,2 Fax: 0131-242-6578 E-mail: John.Iredale@ed.ac.uk With repeated and chronic injury, inflammation is Manuscript received: November 12, 2009. persistent or iterative and the normal wound hea- Manuscript accepted: November 12, 2009. ling response becomes aberrant with excessive depo- 284 Ramachandran P. et al. , 2009; 8 (4): 283-291 sition of ECM, primarily fibrillar collagens I and some of these enzymes may in fact serve to further III.3,4 The principal cellular source of this ECM has enhance HSC activation, it is clear that there is sig- been identified as the activated hepatic stellate cell nificant matrix degrading potential already present (HSC). When quiescent, HSCs reside within the spa- within the fibrotic liver. So what is it that keeps ce of Disse and principally store retinoids. Follo- these enzymes in check during progressive fibrosis? wing injury, HSCs undergo transdifferentiation into Tissue Inhibitors of Metalloproteinases (TIMPs) an activated myofibroblast-like phenotype, associa- are potent inhibitors of MMPs. During activation ted with loss of the retinoid droplets and expression HSCs upregulate TIMP-1 and TIMP-2, even prior to of the filament alpha smooth muscle actin (α- expression of collagen I. This HSC derived TIMP-1, SMA).5-8 They then adopt a number of pro-fibrotic inhibits secreted MMP-2 and MMP-9 activity more features including proliferation, contractility, ECM than 20-fold.22,23 Subsequent studies demonstrated synthesis, secretion of pro-inflammatory mediators elevated levels of TIMP-1 and TIMP-2 in progressive and inhibition of matrix degradation (see below).9 fibrosis in animal models as well as explanted hu- Thus, they are critical in the progression of liver fi- man liver.21,22,24 This has even lead to the proposed brosis. use of TIMP-1 as a serum marker of fibrosis.25 An In addition to HSCs, other sources of hepatic elegant study Yoshiji et al confirmed a functional myofibroblasts have also been identified. Portal role for TIMP-1 in fibrosis progression. By utilising myofibroblasts have been defined as a distinct popu- a transgenic mouse model with liver specific TIMP-1 lation, which may have enhanced significance in overexpression, they demonstrated that no increase chronic cholestatic disease or periportal fibrosis.10-12 in basal fibrosis was seen but following injury the Bone-marrow derived myofibroblasts also contribute TIMP-1 overexpressing mouse had markedly increa- significantly to liver fibrosis. This interesting fin- sed levels of scarring.26 This combination of data ding was demonstrated by studying male patients has lead to the hypothesis that the overall MMP- who had received a liver transplant from a female TIMP balance is critical for fibrosis progression. donor, with recurrent fibrosis in the graft. The au- thors were able to demonstrate that a significant IS HUMAN LIVER proportion of the myofibroblasts in these livers were FIBROSIS REVERSIBLE? Y-chromosome positive, confirming their origin from circulating cells.13 This has subsequently been A careful review of the literature reveals that ane- confirmed in murine models including tangible evi- cdotal evidence for the reversibility of liver fibrosis dence that these cells are functional in vivo.14 Epi- has existed for over 30 years. Subsequently, the de- thelial-mesenchymal transition (EMT), where velopment of effective treatments for chronic hepati- hepatic myofibroblasts arise from differentiation of tis B and hepatitis C lead to clinical trial data with hepatocytes or cholangiocytes, has also been propo- liver biopsies before and after therapy. These studies sed in liver fibrosis,15-17 although the relative func- convincingly demonstrated, for the first time in lar- tional contribution of these cells remains to be ge cohorts of patients, that resolution of fibrosis elucidated. was a real phenomenon.27-29 Similar findings have also been demonstrated in chronic liver diseases of FIBROSIS PROGRESSION RESULTS other aetiologies including alcohol,30 autoimmune FROM A FAILURE IN MATRIX DEGRADATION hepatitis,31,32 chronic biliary obstruction,33 heredita- ry haemochromatosis34 and NAFLD.35 The diversity Matrix metalloproteinases (MMPs) are a group of in the range of conditions represented in this list enzymes capable of degrading different components would suggest that reversibility is potentially gene- of the ECM. Studies in whole liver of both animal ric rather than disease specific and raises hope that models and human cirrhosis have demonstrated an a therapeutic intervention could be effective. increase in MMPs active against a range of ECM proteins.2,18-20 Furthermore, tissue culture studies of MYOFIBROBLAST APOPTOSIS AND isolated HSCs have shown that when activated they MATRIX DEGRADATION ARE CENTRAL can express a plethora of these MMPs. Early during FEATURES OF FIBROSIS RESOLUTION activation, transient upregulation of MMP-13 (co- llagenase-3) and MMP-3 (stromelysin) is seen, follo- Rodent studies of bile duct ligation (BDL) follo- wed by increases in MMP-2 (gelatinase A), MMP-9 wed by bilio-jejunal anastamosis or chronic carbon (gelatinase B) and MMP-14 (MT-MMP).21 Whilst tetrachloride (CCl4) administration followed by cess- 285 Reversibility of liver fibrosis. , 2009; 8 (4): 283-291 ation of dosing are both excellent models to study solution. Cells of the monocyte/macrophage lineage the dynamics of fibrogenesis and spontaneous reso- are central in this. Macrophages are capable of lution. In each of these situations, an established adopting different activation states in response to di- hepatic fibrosis can resolve to near normal liver ar- fferent environmental cues, including pro-and anti- chitecture within 4-6 weeks.36,37 Two key histologi- inflammatory and wound healing phenotypes.46 cal changes are observed during this resolution Zamara and colleagues have demonstrated that mice phase: degradation of fibrotic matrix (see below) and lacking the monocyte chemoattractant protein MCP- a dramatic loss of the activated HSC/myofibroblast 1, have reduced levels of hepatic inflammation follo- population. Closer analysis indicates that the HSCs wing acute injury. 47 Furthermore, inhibition of are lost by the process of programmed cell death, or MCP-1 with a dominant-negative mutant followed apoptosis.36,37 This finding lead to a series of in vitro by chronic hepatic injury with dimethylnitrosamine studies examining the signals regulating HSC apop- in a rat model, clearly shows a reduction in liver tosis.38 These signals include pro-apoptotic soluble macrophage infiltration and protection from fibroge- mediators such as Nerve Growth Factor (NGF) nesis. 48 The seminal work in this field was publis- which can be released from infiltrating inflamma- hed by Duffield, et al.49 Using a transgenic mouse tory cells or regenerating hepatocytes.39,40 Further- model enabling specific depletion of macrophages, more, direct cellular contact between macrophages they were able to mirror the above findings, and activated HSCs has been demonstrated to pro- showing a reduction in fibrosis and myofibroblast mote HSC death.41 Intriguingly, interactions bet- number by depletion during progressive injury with ween the ECM itself and HSCs can modulate CCl4. Intriguingly, depletion during spontaneous re- apoptosis. Binding between collagen-1 and HSCs solution abrogated the improvement in fibrosis, sug- promote activation whereas loss of integrin-media- gesting that macrophages also have a critical ted contact can lead to the HSCs entering the apop- involvement in matrix remodelling. This data totic pathway.42 Similar findings have been showed, for the first time, the existence of functio- demonstrated in vivo by Issa and colleagues, where nally distinct subpopulations of macrophages in the a transgenic mouse expressing a non-degradable liver during injury and recovery. form of collagen-1 showed a failure of spontaneous The mechanism of these macrophage driven fibrosis resolution and a persistence of activated effects has also been the subject of scrutiny (Figure HSCs in the scar following chronic CCL 4.43 Taken 1). Further analysis has revealed that following in- together, this evidence highlights the critical impor- jury, there is a contribution to the hepatic macro- tance of the myobroblast-matrix interaction in fibro- phage pool from both liver resident Kupffer cells as sis resolution-ECM degradation will result in well as circulating bone-marrow derived cells.49 In reduced survival/increased apoptotic signals to the addition, review of the histology shows that the ma- HSCs, which in turn will undergo cell death leading crophages are located closely associated with the he- to a reduction in ECM production. patic scar, again confirming their central role.49,50 Clearly, matrix degradation is required in addi- In vitro studies have demonstrated that macropha- tion to HSC apoptosis for adequate fibrosis resolu- ges are capable of promoting HSC activation via tion. Once again studies in animal models have TGF-β production51 and increasing PDGF receptor given us critical clues into this process. Studying a expression,52 potentially explaining their profibrotic series of timepoints during fibrosis resolution indi- role. Conversely, they can also promote HSC apop- cates a significant reduction in the levels of TIMP-1 tosis via expression of molecules such as TRAIL41 or and TIMP-236,44 with an associated increase in ove- MMP-9. 42 Moreover, Fallowfield and colleagues rall collagenase activity due to a switch in the showed that scar-associtated macrophages in the li- MMP-TIMP balance, resulting in net ECM degrada- ver are the predominant source of MMP-13, the ma- tion.44 Furthermore, TIMP-1 in itself is a pro-survi- jor rodent interstitial collagenase, and that val signal for HSCs,45 highlighting the importance MMP-13-deficient mice fail to resolve the scar.50 of this reduction in concentration during resolution. These findings raise the possibility of manipulating macrophages as a therapeutic modality. MACROPHAGES IN The relative contribution of other cells of the in- FIBROSIS RESOLUTION nate and adaptive immune system in liver fibrosis is less well established, although a complex interplay Inflammatory cells, both resident and recruited, clearly exists.1 Inhibition of neutrophil recruitment, also have a key role to play in both fibrosis and re- using either anti-neutrophil serum53 or CXCR2 286 Ramachandran P. et al. , 2009; 8 (4): 283-291 PROGRESIVE FIBROSIS Quiescent HSC Activated HSC HSC activation HSC proliferation Figure 1. The central role of Scar Associated Macrophages (SAMs) in TG-β PDGF liver fibrosis progression and resolu- tion. During progressive liber fibro- sis, SAMs can produce factors enhancing hepatic stellate cell (HSC) activation (e.g. TGF-β) and prolife- ration (e.g. Platelet Derived Growth Factor (PDGF), leading to a popula- Scar Associated Macrophage tion of matrix-producing activated myofibroblasts. During the resolu- tion phase, SAMs enhance loss of TRAIL MMP-13 ECM HSCs by apoptosis (e.g. via expres- Degradation sion of death ligands such as TRAIL) and have a direct effect on extrace- HSC apoptosis llular matrix (ECM) degradation by expression of matrix metalloprotei- Loss of HSC-ECM nases such as MMP-13. ECM degra- interactions dtion further enhaces HSC apoptosis RESOLUTION by loss of survival signals mediated by HSC-ESC interactions. knockout mice54 seems to have minimal effect on fi- ced fibrosis, but also encompasses abnormal hepato- brosis. In a B Cell deficient mouse, however, there cyte regeneration, architectural disturbance and is reduced liver fibrosis in response to chronic CCL4 vascular changes. Once again experimental models administration, while no difference was seen in T have given us insight into this key question. Issa et cell deficient mice by this group.55 A proposed me- al induced cirrhosis in rats by 12 weeks of CCL4 ad- chanism for this finding is that there may be enhan- ministration, followed by a protracted recovery pe- ced clearance of apoptotic hepatocytes by riod of up to 1 year.44 After 12 weeks of injury, macrophages.56 On the other hand, B cell deficient micronodules were clearly visible surrounded by mice show increased hepatic fibrosis in response to dense mature fibrotic septa. Following recovery, sig- chronic schistosoma infection57 whilst adoptive nificant remodelling was observed, primarily in the transfer of CD8+ve T Cells from CCl4 treated mice freshly formed fibrosis immediately adjacent to into immunodeficient SCID mice causes activation the nodules. The more mature scars, however, failed of HSCs.58 Such findings demonstrate the difficulty to resolve completely even after a prolonged period. in unpicking the key mechanisms involved and it is By comparing the reversible with the potentially certain that disease- or model-specific factors will be irreversible scars, some of the features leading to at play. There also remains a lack of mechanistic irreversibility could be defined. Principally, the data on the relative contribution of these cell types irreversible septae contained greater quantities of during the resolution from fibrosis. the ECM component elastin. Furthermore, tissue transglutaminase mediated matrix cross-linking WHAT RENDERS could be demonstrated within the irreversible FIBROSIS IRREVERSIBLE? scars.44 This would suggest the possibility that the more mature scar contains cross-linked matrix ren- Having described some of the evidence confirming dering it resistant to proteolytic degradation, simi- the reversibility of liver fibrosis in human and ani- lar to fibrosis in other organ systems.59 The other mal models and the key mechanistic events in this distinguishing feature of the irreversible scars was process, what remains uncertain is whether advan- that they were relatively paucicellular, containing ced cirrhosis can return to normal liver architecture fewer active myofibroblasts and macrophages than or whether there is a point of no return. It is im- their reversible counterparts.44 This again could be portant to note that cirrhosis is not simply advan- critical, the implication being that the absence of ce- 287 Reversibility of liver fibrosis. , 2009; 8 (4): 283-291 lls within the scar leads to reduced delivery of ma- the management of type 2 diabetes and the in vitro trix degrading enzymes to the area. findings tally nicely with the recently published cli- Similar evidence in human liver disease is still nical trials in NAFLD, demonstrating histological lacking. However, a study by Wanless et al looking improvement in the liver following treatment.65-67 at explanted cirrhotic human livers, did indicate Angiotensin II also has an activating effect on partial regression even from advanced micronodular HSCs, probably mediated via TGF-β expression, and cirrhosis. 60 Strikingly, the histological patterns blockade of the renin-angiotensin system seems to were remarkably similar to that described in the rat ameliorate experimental liver fibrosis68-70 as well as model,44 suggesting common mechanisms might be potentially having benefits in human disease.71 This prevalent. correlates well with the reported effects on cardiac and renal fibrosis.72,73 TARGETED THERAPIES Given critical nature of HSC apoptosis in fibrosis IN FIBROGENESIS AND RESOLUTION resolution, several authors have attempted to stimu- late this as an anti-fibrotic therapy. As discussed As described there is extensive data on the rever- above activated HSCs respond to a variety of apop- sibility of fibrosis and at least in part of cirrhosis. totic signals and express several death receptors in- This raises hopes that an effective anti-fibrotic the- cluding p75, FAS and TNF-α, in addition to the rapy can soon be translated into the clinic. As our critical interaction with ECM.2 Attempts to specifi- understanding of the pathogenic mechanisms has cally target this have been made, but are limited by evolved, numerous researchers have attempted to potential off target effects due to the pleotropic natu- target different fibrogenic pathways. A review of the re of these molecules. An observation that activated literature would reveal hundreds of such anti-fibro- HSCs persistently express high levels of NF-κB, a tic interventions in animal models. These can pro-inflammatory transcription factor, and that this broadly be divided into 3 main categories: confers protection from HSC apoptosis has lead to additional strategies to target this process.74 Sul- 1. Targeting the injurious stimulus or the inflam- phasalazine, a widely used anti-inflammatory drug, matory process ultimately leading to myofibro- has been shown to induce HSC apoptosis in vitro blast activation and ECM deposition. and in vivo via an NF-κB dependent mechanism and 2. Reducing myofibroblast activation and promo- accelerates fibrosis resolution in a rat model.75 Glio- ting myofibroblast loss by apoptosis. toxin, a fungal metabolite, also induces HSC apopto- 3. Modulating the MMP-TIMP balance to favour sis, at least in part due to NF-κB inhibition, and ECM degradation. thus has anti-fibrotic effects.76 This also had off tar- get effects causing depletion of hepatic macrophages. Given the importance of the resolution phase be- To circumvent this problem authors have used an cause patients invariably present to medical atten- antibody raised against myofibroblasts to target the tion with established disease, this review will now gliotoxin to HSCs and gain a much more specific focus on the latter 2 aspects. effect.77 This certainly represents a significant ad- Various molecules have been shown to reduce the vance, and similar methods may be applied more pro-fibrotic nature of HSCs. One particular example widely in the future. is relaxin. This hormone is normally produced by Having described the importance of the MMP- the corpus luteum during the 3rd trimester of preg- TIMP balance in the progression and resolution of nancy and acts to soften the symphysis pubis. Stu- fibrosis, not surprisingly various studies have at- dies using relaxin on activated HSCs have shown tempted to target this. Initially attempts were made that it modulates TIMP and collagen expression in to increase hepatic MMP levels. Siller-Lopez and co- vitro and that it is anti-fibrotic in vivo,61 suggesting lleagues administered adenovirus causing hepatic that recombinant relaxin or synthetic analogues overexpression of MMP-8 (neutrophil collagenase) may have a therapeutic role. The peroxisome prolife- and showed that this significantly reduced fibrosis rator-activated receptors (PPAR) also have an im- following BDL in rats.78 TIMP inhibition as a thera- portant role in regulating HSC behaviour. During peutic avenue has also received some attention. A HSC activation a marked reduction of PPARγ expre- TIMP-1 neutralising antibody resulted in reduced ssion is seen and synthetic PPARγ agonists reduce progression in a rat CCL4 model.79 Roeb and collea- HSC proliferation, chemotaxis and collagen-1 expre- gues used a particularly novel method of TIMP inhi- ssion.62-64 These drugs are already widely used in bition. They developed a series of MMP-9 mutants, 288 Ramachandran P. et al. , 2009; 8 (4): 283-291 which lacked MMP gelatinolytic activity, but retai- renal injury, using macrophages either stimulated ned their TIMP binding properties. These mutants with cytokines85 or virally transduced to express were able to inhibit TIMP-1 effects in vitro and in anti-inflammatory cytokines86,87 prior to adoptive vivo and reduced fibrosis in a murine model.80,81 transfer. Studies such as these are still lacking in li- This strategy would once again have the advantage ver fibrosis models. of minimising off-target effects, by specifically ha- ving action in areas where TIMP-1 would be upre- CONCLUSION gulated during fibrogenesis. Clearly a full discussion of the myriad of poten- Our understanding of the pathogenesis of hepatic tial anti-fibrotic therapies that have been tried in ex- fibrosis has progressed exponentially in the past 30 perimental models is beyond the scope of this years. In particular, we now have cogent and con- article. However, what is clear from the selected sistent evidence that fibrosis (and to a lesser degree examples, is that our greater understanding of the advanced cirrhosis) is potentially reversible in hu- pathogenesis has enabled specific and targetted the- mans and animal models, and have an insight into rapies to be developed. The challenge remains in the the critical molecular and cellular pathways invol- translation of these to the clinical setting, in parti- ved. We have also gained knowledge of some of the cular the avoidance of off-target effects which could features preventing resolution of mature scars. Uti- potentially cause adverse events in humans. lising these findings, several novel therapies have been trialled in reproducible animal models of liver THE POTENTIAL OF CELL THERAPY disease. What remains elusive is effective translation into the human setting. In the future, cellular the- As discussed above, a cardinal feature of the irre- rapies or treatments targeting multiple points in versible scar is its hypocellularity. Many groups are the complex fibrogenic pathway may help to bridge now exploring the use of cellular therapy for liver fi- this gap. brosis. This could potentially have the benefit of see- ding the irreversible scar, enabling modification of ABBREVIATIONS the local microenvironment and possible degrada- tion. A variety of cell types have been used for this ECM: Extracellular matrix. purpose, with a focus on a stem cell fractions. HSC: Hepatic stellate cell. Sakaida and colleagues, for example, used adoptive MMP: Matrix metalloproteinase. transfer of GFP-expressing bone marrow cell frac- TIMP: Tissue Inhibitor of metalloproteinases. tion into mice during progressive CCL4 induced in- TRAIL: TNF-related apoptosis inducing ligand. jury. They were able to demonstrate a reduction in fibrosis, and also that the injected cells localised FUNDING around the scars and co-expressed MMP-9.82 The implication of this is that they may become pro-reso- Prakash Ramachandran is funded by The Wellco- lution scar-associated macrophages. It has also been me Trust. proposed that injected stem cell fractions could diffe- John P Iredale is consultan for GE Healhcare and rentiate and contribute to the regenerating hepato- is funded by The Wellcome Trust, Medical Research cyte pool, although this remains controversial.83 Council United Kingdom and the Childrens Liver Clinical trials of cell therapy have been conducted- Disease Foundation. whilst these are generally small, non-randomised and do not show unequivocal benefit, they do offer REFERENCES some hope for the future.84 A criticism of the cell therapy strategy would be 1. Henderson NC, Iredale JP. Liver fibrosis: cellular mechanis- ms of progression and resolution. Clin Sci (Lond) 2007; that following adoptive transfer, the cells will en- 112(5): 265-80. counter the existing microenvironment in the liver, 2. Iredale J. 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