senescence of activated stellate cells: not just early retirement

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HEPATOLOGY ELSEWHERE EDITORS Kris Kowdley, Seattle, WA Geoffrey McCaughan, Newtown, Australia Christian Trautwein, Aachen, Germany Senescence of Activated Stellate Cells: Not Just Early Retirement Krizhanovsky V, Yon M, Dickins RA, Hearn S, Simon J, Miething C, et al. Senescence of activated stellate cells limits liver fibrosis. Cell 2008;134:657-667. (Reprinted with permission.) Abstract Cellular senescence acts as a potent mechanism of tumor sup- pression; however, its functional contribution to noncancer pathologies has not been examined. Here we show that senes- cent cells accumulate in murine livers treated to produce fibro- sis, a precursor pathology to cirrhosis. The senescent cells are derived primarily from activated hepatic stellate cells, which initially proliferate in response to liver damage and produce the extracellular matrix deposited in the fibrotic scar. In mice lack- ing key senescence regulators, stellate cells continue to prolif- erate, leading to excessive liver fibrosis. Furthermore, senescent activated stellate cells exhibit gene expression profile consistent with cell-cycle exit, reduced secretion of extracellular matrix components, enhanced secretion of extracellular matrix-de- grading enzymes, and enhanced immune surveillance. Accord- ingly natural killer cells preferentially kill senescent activated stellate cells in vitro and in vivo, thereby facilitating the reso- lution of fibrosis. Therefore, the senescence program limits the fibrogenic response to acute tissue damage. Comment Cirrhosis, the architectural disruption of the liver with abnormal hepatocyte regeneration and extensive fibrosis, re- sults from relentless cycles of inflammation and repair initi- ated and perpetuated by diverse causes including chronic viral infection, metabolic disorders, toxic damage, and para- sitism. During hepatic injury, hepatocyte damage and the consequent inflammation drive the transdifferentiation of hepatic stellate cells (HSCs) to become activated myofibro- blast-like cells. In this activated phenotype, HSCs are re- sponsible for secretion of the fibrotic matrix rich in interstitial collagens. 1 Stellate cells, and other myofibroblast lineages present in injury and fibrosis, are also subject to intense proliferative stimuli, including platelet-derived growth factor. However, despite these stimuli, and after years or even decades of cycles of inflammation and repair, the numbers of myofibroblasts remain regulated. Indeed, dys- regulated proliferation in the form of cancer is seen in the hepatocyte or hepatocyte progenitor component of the cir- rhotic liver but not in the myofibroblast component. Although much is known about the stimuli that pro- mote HSC activation and proliferation, we actually know relatively little about the mechanisms that limit or reduce myofibroblast numbers in the liver in vivo. Previous work has demonstrated that resolution of liver fibrosis, which may occur even in comparatively advanced fibrosis and early cirrhosis, is accompanied by a marked reduction in the number of HSCs/myofibroblasts—a phenomenon mediated by apoptosis. 2 Additionally, liver natural killer (NK) cells have been shown to selectively delete the HSC/ myofibroblast pool. 3 Previous studies in tissue culture models have also suggested that human (Hu) HSCs/myo- fibroblasts may be susceptible to the development of rep- licative senescence after multiple cycles of proliferation. 4,5 The process of senescence was first described as a state of terminal proliferative exhaustion in fibroblast cell culture. 6 Subsequently, it has been shown to be mediated by progres- sive telomere shortening and activation of a DNA damage response. Senescence has also been described to occur after supraphysiological stimulation of mitogenic pathways by oncogenic transformation of cells in culture. 7 The pheno- type of senescent cells is characterized by a terminal cell cycle arrest, expression of beta galactosidase, and induction of p53, p21, and p16. The p53 and p16 proteins seem to play a pivotal role in the process of senescence. Studies in other organ systems suggest that the development of senescence may prevent the progression to cancer. 8 Given this background, the paper published by Krizhanovsky et al. 9 in Cell represents an exciting de- velopment in our understanding of the mechanisms governing HSC/myofibroblast numbers in diseased liver tissue. Using beta-Gal staining, p53, p21, p16, and Hmga 1 expression to verify the senescent pheno- type and by co-localizing these markers with alpha smooth muscle actin and desmin, Krizhanovsky and co-workers have identified for the first time evidence of senescent myofibroblasts in areas of fibrosis in CCl 4 - injured mouse liver. By using p53 and p16 knockout mice, they go on to show mechanistically that animals with a blocked senescence programme develop more severe fibrosis after a given duration of injury and that the fibrosis is slower to resolve spontaneously. The number of alpha smooth muscle actin–positive myofi- broblasts is markedly up-regulated in p53 and p16/p53 double-knockout mice during the development of fi- brosis and fibrosis resolution. This suggests that these 1045

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Page 1: Senescence of activated stellate cells: Not just early retirement

HEPATOLOGY ELSEWHERE EDITORSKris Kowdley, Seattle, WAGeoffrey McCaughan, Newtown, AustraliaChristian Trautwein, Aachen, Germany

Senescence of Activated Stellate Cells: NotJust Early Retirement

Krizhanovsky V, Yon M, Dickins RA, Hearn S, Simon J,Miething C, et al. Senescence of activated stellate cellslimits liver fibrosis. Cell 2008;134:657-667. (Reprintedwith permission.)

AbstractCellular senescence acts as a potent mechanism of tumor sup-pression; however, its functional contribution to noncancerpathologies has not been examined. Here we show that senes-cent cells accumulate in murine livers treated to produce fibro-sis, a precursor pathology to cirrhosis. The senescent cells arederived primarily from activated hepatic stellate cells, whichinitially proliferate in response to liver damage and produce theextracellular matrix deposited in the fibrotic scar. In mice lack-ing key senescence regulators, stellate cells continue to prolif-erate, leading to excessive liver fibrosis. Furthermore, senescentactivated stellate cells exhibit gene expression profile consistentwith cell-cycle exit, reduced secretion of extracellular matrixcomponents, enhanced secretion of extracellular matrix-de-grading enzymes, and enhanced immune surveillance. Accord-ingly natural killer cells preferentially kill senescent activatedstellate cells in vitro and in vivo, thereby facilitating the reso-lution of fibrosis. Therefore, the senescence program limits thefibrogenic response to acute tissue damage.

CommentCirrhosis, the architectural disruption of the liver with

abnormal hepatocyte regeneration and extensive fibrosis, re-sults from relentless cycles of inflammation and repair initi-ated and perpetuated by diverse causes including chronicviral infection, metabolic disorders, toxic damage, and para-sitism. During hepatic injury, hepatocyte damage and theconsequent inflammation drive the transdifferentiation ofhepatic stellate cells (HSCs) to become activated myofibro-blast-like cells. In this activated phenotype, HSCs are re-sponsible for secretion of the fibrotic matrix rich ininterstitial collagens.1 Stellate cells, and other myofibroblastlineages present in injury and fibrosis, are also subject tointense proliferative stimuli, including platelet-derivedgrowth factor. However, despite these stimuli, and after yearsor even decades of cycles of inflammation and repair, thenumbers of myofibroblasts remain regulated. Indeed, dys-regulated proliferation in the form of cancer is seen in thehepatocyte or hepatocyte progenitor component of the cir-rhotic liver but not in the myofibroblast component.

Although much is known about the stimuli that pro-mote HSC activation and proliferation, we actually knowrelatively little about the mechanisms that limit or reducemyofibroblast numbers in the liver in vivo. Previous workhas demonstrated that resolution of liver fibrosis, whichmay occur even in comparatively advanced fibrosis andearly cirrhosis, is accompanied by a marked reduction inthe number of HSCs/myofibroblasts—a phenomenonmediated by apoptosis.2 Additionally, liver natural killer(NK) cells have been shown to selectively delete the HSC/myofibroblast pool.3 Previous studies in tissue culturemodels have also suggested that human (Hu) HSCs/myo-fibroblasts may be susceptible to the development of rep-licative senescence after multiple cycles of proliferation.4,5

The process of senescence was first described as a state ofterminal proliferative exhaustion in fibroblast cell culture.6

Subsequently, it has been shown to be mediated by progres-sive telomere shortening and activation of a DNA damageresponse. Senescence has also been described to occur aftersupraphysiological stimulation of mitogenic pathways byoncogenic transformation of cells in culture.7 The pheno-type of senescent cells is characterized by a terminal cell cyclearrest, expression of beta galactosidase, and induction of p53,p21, and p16. The p53 and p16 proteins seem to play apivotal role in the process of senescence. Studies in otherorgan systems suggest that the development of senescencemay prevent the progression to cancer.8

Given this background, the paper published byKrizhanovsky et al.9 in Cell represents an exciting de-velopment in our understanding of the mechanismsgoverning HSC/myofibroblast numbers in diseasedliver tissue. Using beta-Gal staining, p53, p21, p16,and Hmga 1 expression to verify the senescent pheno-type and by co-localizing these markers with alphasmooth muscle actin and desmin, Krizhanovsky andco-workers have identified for the first time evidence ofsenescent myofibroblasts in areas of fibrosis in CCl4-injured mouse liver. By using p53 and p16 knockoutmice, they go on to show mechanistically that animalswith a blocked senescence programme develop moresevere fibrosis after a given duration of injury and thatthe fibrosis is slower to resolve spontaneously. Thenumber of alpha smooth muscle actin–positive myofi-broblasts is markedly up-regulated in p53 and p16/p53double-knockout mice during the development of fi-brosis and fibrosis resolution. This suggests that these

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Page 2: Senescence of activated stellate cells: Not just early retirement

proteins, which are known to regulate the senescent pheno-type, are key players in regulating the controlled proliferationand loss of HSC/myofibroblasts. To confirm that the ob-served effects were specific to HSCs, the authors went on todevelop transgenic mice with a silencing RNA construct un-der the control of the glial fibrillary acidic protein promoter,thereby blocking the p53-induced senescence response onlyin the HSC population of the liver. These elegant in vivostudies were accompanied by messenger RNA profiling intissue culture models of DNA-damaged senescent HuHSCand a fibroblast cell line. Here the authors observed a discretesenescence phenotype characterized by down-regulation ofmatrix production and an up-regulation of matrix-degradingenzymes. Furthermore, these senescent cells strongly up-reg-ulated immune stimulatory cytokines and NK cell–activat-ing surface markers. Given that NK cells have previouslybeen shown to mediate deletion of HSCs,3 Krizhanovskyand colleagues went on to further characterize the role of NKcells in the regression of liver fibrosis. Depletion of NK cellsby specific antibodies markedly delayed resolution, whereasstimulation of the innate immune response with polyi-nosinic:polycytidylic acid speeded up resolution reflected ina higher clearance of alpha smooth muscle actin–positivecells from the fibrotic scar.

Taken together, these data point to senescence as a newstage in the life cycle of an activated HSC/myofibroblast(Fig. 1). Whereas the data generated with the murine modelsare compelling, the confirmatory experiments using humancells in tissue culture are important. Senescence has beendemonstrated previously in culture-activated HuHSC, and acomplex phenotype is emerging characterized by reduced

matrix synthesis with an enhanced inflammatory pheno-type.4 As noted, enhanced immune surveillance resultingfrom these changes may lead to NK cells preferentially killingsenescent activated HSC/myofibroblasts. Other workershave defined that senescent HuHSC demonstrate a lowerBCL2-expressing phenotype compared with activated pro-liferating HSC. This might explain the higher susceptibilityto apoptosis of senescent HSCs.5 These observations in hu-man cells are critical, because human cells lack telomeraseand after serial passage in culture will develop shortened telo-meres and could be considered to develop a classical replica-tive senescence as originally defined. In contrast, the in vivowork presented in the study by Krizhanovsky et al. is under-taken in mice—a mammal that expresses telomerase in awide range of cell lineages. Indeed, manipulation of telom-erase expression has been required to achieve replicative se-nescence in murine hepatic cell lines. This suggests that thesenescence program described by Krizhanovsky et al. mayrepresent a new and specific regulatory mechanism for fibro-proliferative disorders that is not dependent on telomere ero-sion and replicative senescence.

As with every groundbreaking study, many criticalquestions arise. A key question posed by this report is howthe senescence program is initiated and regulated inHSCs/myofibroblasts, particularly if it may occur in theabsence of shortened telomeres. The authors discuss thepossibility of replicative senescence but, probably cor-rectly, conclude that on the basis of the species used andthe short period of injury this is an unlikely underlyingmechanism. A more appealing and intriguing concept isthat overstimulation of the cells by cytokines and growth

Fig. 1. The life cycle of a hepatic stellatecell in liver fibrosis.

1046 HEPATOLOGY ELSEWHERE HEPATOLOGY, March 2009

Page 3: Senescence of activated stellate cells: Not just early retirement

factors leads to a state comparable to mitogenic over stim-ulation observed in oncogene transformed cells. Indeed,in this study an active Akt pathway is observed in bothhighly activated and senescent HSC, suggesting activa-tion of comparable mechanisms.

The link between the senescence phenotype describedby Krizhanovsky et al. and apoptosis of HSC also remainsto be resolved. It is of course possible that apoptosis, oc-curring as a result of stimulation of death receptors byspecific ligands or withdrawal of survival factors, may oc-cur in parallel with NK cell deletion of activated HSCs/myofibroblasts during resolution of fibrosis. Additionally,of course, deletion of activated HSC/myofibroblast byNK cells may induce an apoptotic pathway. The resis-tance of HuHSCs to classical apoptotic stimuli and thehigh expression of BCL2 by such cells suggest that withfurther study we may define differences in cells that haveundergone replicative senescence as opposed to the senes-cence program described by Krizhanovsky et al. occurringin response to promitogenic stimuli.

A furtherquestion ishowrelevant theobservationsmade inamurine model are to the clinical situation and the relative role ofsenescence in the development and progression of human liverfibrosis. Here interpretation becomes extremely complex. Se-nescent hepatocytes have been identified in the livers of patientswith cirrhosis and are associated with shortened telomeres.10

The senescence program also might be involved in the silencingof premalignant hepatocytes or hepatic progenitor cells, therebypreventing the emergenceofhepatocellular carcinoma.11 More-over, in a seminal paper published in Science in 2000, DePinhoand colleagues demonstrated accelerated fibrosis in mice withdeficient telomerase, pointing strongly to a profibrotic effect ofreplicative senescence in hepatocytes.12 However, livers are notsimply organs consisting of hepatocytes and HSCs/myofibro-blasts. Senescence has also been reported in chronic bile ductinflammation in the setting of primary biliary cirrhosis and pri-mary sclerosing cholangitis.13 Here evidence of telomere short-ening is associated with senescent biliary epithelial cells and areduction in the number of small bile ducts. Finally, there isincreasing evidence that senescence may occur in the immunesystem during chronic viral infection, and work published inabstract formsuggests that lymphocytesenescencealsomaypro-mote the development of fibrosis.14 Thus, in the adult humanwith active chronic disease, the net effect of replicative senes-cence, and senescence induced as described by Krizhanovsky etal., on the organ remains to be defined.

In summary, the finding of senescence in HSCs during thedevelopment of fibrosis and resolution and the elegant link be-tween this phenotype and deletion by NK cells have identified anovelmodel for the regulationofHSC/myofibroblast cellnum-bers. Moreover, this phenomenon may prove to be importantfor fibroproliferative disorders elsewhere in the body. That the

observed senescence program occurs in cells that are unlikely tohave significantly shortened telomeres suggests that HSC/myo-fibroblasts may be susceptible to a mitogen-induced senescenceprogrammethatmaybeanalogous to that seen incells subject tooncogenic stimulation. Krizhanovsky et al. have made a series ofcritical observations that significantly enhance our understand-ingof thepathogenesisoffibrosis and thatmayprove importantin the design of therapeutic approaches in the future.

JORG SCHRADER

JONATHAN FALLOWFIELD

JOHN P. IREDALE

MRC Centre for Inflammation ResearchUniversity of EdinburghEdinburgh, UK

References1. Friedman SL. Mechanisms of hepatic fibrogenesis. Gastroenterology

2008;134:1655-1669.2. Iredale JP, Benyon RC, Pickering J, McCullen M, Northrop M, Pawley S,

et al. Mechanisms of spontaneous resolution of rat liver fibrosis: hepaticstellate cell apoptosis and reduced hepatic expression of metalloproteinaseinhibitors. J Clin Invest 1998;102:538-549.

3. Radaeva S, Sun R, Jaruga B, Nguyen VT, Tian Z, Gao B. Natural killercells ameliorate liver fibrosis by killing activated stellate cells in NKG2D-dependent and tumor necrosis factor-related apoptosis-inducing ligand-dependent manners. Gastroenterology 2006;130:435-452.

4. Schnabl B, Purbeck CA, Choi YH, Hagedorn CH, Brenner DA. Replica-tive senescence of activated human hepatic stellate cells is accompanied bya pronounced inflammatory but less fibrogenic phenotype. HEPATOLOGY

2003;37:653-664.5. Novo E, Marra F, Zamara E, Valfre di Bonzo L, Monitillo L, Cannito S, et

al. Overexpression of Bcl-2 by activated human hepatic stellate cells: resis-tance to apoptosis as a mechanism of progressive hepatic fibrogenesis inhumans. Gut 2006;55:1174-1182.

6. Hayflick L. The limited in vitro lifetime of human diploid cell strains. ExpCell Res 1965;37:614-636.

7. Campisi J, d’Adda di Fagagna F. Cellular senescence: when bad thingshappen to good cells. Nat Rev Mol Cell Biol 2007;8:729-740.

8. Collado M, Blasco MA, Serrano M. Cellular senescence in cancer andaging. Cell 2007;130:223-233.

9. Krizhanovsky V, Yon M, Dickins RA, Hearn S, Simon J, Miething C, et al.Senescence of activated stellate cells limits liver fibrosis. Cell 2008;134:657-667.

10. Wiemann SU, Satyanarayana A, Tsahuridu M, Tillmann HL, Zender L,Klempnauer J, et al. Hepatocyte telomere shortening and senescence aregeneral markers of human liver cirrhosis. FASEB J 2002;16:935-942.

11. Xue W, Zender L, Miething C, Dickins RA, Hernando E, KrizhanovskyV, et al. Senescence and tumour clearance is triggered by p53 restoration inmurine liver carcinomas. Nature 2007;445:656-660.

12. Rudolph KL, Chang S, Millard M, Schreiber-Agus N, DePinho RA. In-hibition of experimental liver cirrhosis in mice by telomerase gene delivery.Science 2000;287:1253-1258.

13. Sasaki M, Ikeda H, Yamaguchi J, Nakada S, Nakanuma Y. Telomereshortening in the damaged small bile ducts in primary biliary cirrhosisreflects ongoing cellular senescence. HEPATOLOGY 2008;48:186-195.

14. Hoare M, Gelson W, Das A, Fletcher JM, Davies S, Curran MD, et al.Immune senescence, independent of age, informs the clinical outcome ofchronic hepatitis C virus (HCV) infection [Abstract]. HEPATOLOGY 2008;48(Suppl):780A–781A.

Copyright © 2009 by the American Association for the Study of Liver Diseases.Published online in Wiley InterScience (www.interscience.wiley.com).DOI 10.1002/hep.22832Potential conflict of interest: Nothing to report.

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