REVIEWFunctional Role of Cellular Senescence in BiliaryInjury

Q22 Luke Meng,*yz Morgan Quezada,y Phillip Levine,yx Yuyan Han,*y Kelly McDaniel,*yx Tianhao Zhou,*y Emily Lin,*y

Shannon Glaser,*y Fanyin Meng,*yx Heather Francis,*yx and Gianfranco Alpini*y

Q1Q2Q3

From Research, Central Texas Veterans Health Care System,* Temple; the Department of Medicine,y Digestive Disease Research Center, Scott & WhiteHealthcare, Texas A&M Health Science Center, College of Medicine, Baylor Scott & White Health, Temple; the University of Texas Southwestern MedicalCenter,z Dallas; and Academic Operations,x Scott & White Memorial Hospital, Temple, Texas

Accepted for publicationOctober 28, 2014.

Address correspondence toGianfranco Alpini, Ph.D., Scott& White Digestive DiseaseResearch Center, Central TexasVeterans University HealthCare System, Texas A&MHealth Science Center, Olin E.Teague Medical Center, 1901 SFirst St, Bldg 205, 1R60,Temple, TX 76504. E-mail:galpini@tamu.edu.

Cellular senescence is a state of irreversible cell cycle arrest that has been involved in manygastrointestinal diseases, including human cholestatic liver disorders. Senescence may play a rolein biliary atresia, primary sclerosing cholangitis, cellular rejection, and primary biliary cirrhosis,four liver diseases affecting cholangiocytes and the biliary system. In this review, we examineproposed mechanisms of senescence-related biliary diseases, including hypotheses associated withthe senescence-associated phenotype, induction of senescence in nearby cells, and the depletion ofstem cell subpopulations. Current evidence for the molecular mechanisms of senescence in thepreviously mentioned diseases is discussed in detail, with attention to recent advances on the roleof pathways associated with senescence-associated phenotype, stress-induced senescence, telo-mere dysfunction, and autophagy. (Am J Pathol 2015, -: 1e8; http://dx.doi.org/10.1016/

j.ajpath.2014.10.027)

CellularQ6 senescence is a state of irreversible growtharrest in the G1 phase of the cell cycle.1,2 Telomereshortening, double-stranded DNA damage, inflamma-tion, and other forms of cell stress all function asstimuli for cell senescence. Although traditionallycellular senescence has been viewed as a protectiveresponse to cellular injury or disruption, the study ofthe senescence-associated secretory phenotype (SASP)has become increasingly implicated in the pathogenesisof hepatobiliary disease. Specifically, senescence hasbeen hypothesized to play a prominent role in chol-angiopathies, including primary biliary sclerosis [orprimary sclerosing cholangitis (PSC)], primary biliarycholangitis (PBC), cellular rejection (CR), and biliaryatresia (BA). Over time, the SASP may provideproinflammatory or other adverse stimulus to hep-atobiliary stem/progenitor cells, leading to the observeddisease phenotypes.3e5 This review summarizes currentevidence for the molecular mechanisms of cellularsenescence in the pathogenesis of select biliarydiseases.

Cellular Senescence

First observed in 1965, human fibroblasts were noted tohave a limited ability to replicate in culture.6 Senescentcells are metabolically active cells that often remain in situbut no longer maintain proliferative activity. This devel-opment is associated with morphological changes causingthe cells to appear large and flat with characteristic nuclearchanges, including vacuolation.7e10 Two main tumor-suppressor pathways, p53 and p16INK4a/pRB, have beenshown to regulate senescence responses.11 The expressionof associated cell cycle inhibitors, including p15INK4B,p16INK4a, p21WAF1/Cip1, p53, and increased senescence-associated b-galactosidase (SA-b-GAL) activity, is

Supported in part by NIH R01 grants DK062975, DK054811, andDK07698 (G.A., S.G., and F.M Q4.), the Department of Veteran’s AffairsMerit Review Awards (G.A., S.G., and F.M.), the VA Q5CD-2 Award (H.F.),and the PSC Foundation grant (H.F.).L.M., M.Q., and P.L. contributed equally to this work.Disclosures: None declared.

Copyright ª 2014 American Society for Investigative Pathology.Published by Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.ajpath.2014.10.027

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commonly observed. In addition, the production ofsenescence-associated heterochromatin foci and DNA damageresponse proteins have all been used to aid in the identificationof senescent cells in association with the absence of replicativemarkers (Ki-67 or thymidine analogue bromodeoxyuridinein vitro).12 Overall, senescence has been shown to causedifferent effects in different tissues and cell types (Table 1½T1"½T1" ).Correlating expression of these markers with disease states hasdemonstrated the widespread prevalence of the senescentstates in hepatobiliary diseases.

Replicative Senescence

Multiple effectors have been identified leading to the devel-opment of senescence. Classically, replicative senescence de-velops from telomere erosion associated with the accumulationof replication cycles (aging) in the absence of corrective telo-merase (Figure 1½F1"½F1" ). The process of telomere erosion elicits aDNA damage response, which acts to induce the expression ofg-H2Ax (a phosphorylated histone variant of H2Ax) and theDNA damage response proteins p53-binding protein 1, nibrin,

and mediator of DNA damage checkpoint protein 1. DNAdamage kinases ataxia telangiectasia mutated and ataxia tel-angiectasia and Rad3-related protein are subsequently acti-vated and, in turn, activate checkpoint kinases 1 and 2.20,21 Theresultant signal cascade leads to differential expression of p53isoforms, and has been linked directly to senescent pheno-types, although the process, including breakpoints betweenapoptosis and senescence, remains incompletely understood,with some role of NF-kB in its regulation.22,23 In cholestaticliver disease, the role of age-related telomere erosion does notseem prominent. In a study of telomeres using quantitativefluorescent in situ hybridization (Q-FISH) and confirmed bySouthern blot analysis from 73 normal liver samples, chol-angiocytes and hepatocytes were shown to maintain theirtelomere lengths independent of age with only Kupffer andstellate cells, demonstrating age-related attrition.24

Premature Senescence

Numerous other mechanisms independent of replicativesenescence have been identified as pathways of so-called

Table 1 Summary of Effects of the Consequences on Various Cell Types

Cell type Consequences of senescence*

Hepatocytes Unclear, but senescence correlated with fibrosis stage

13

Cholangiocytes Unclear, but senescence correlated with Banff grade in acute rejection

14e16

Hepatic stellate cells Ameliorate fibrosis during acute liver damage via secretion of metalloproteinases

17

Retinal pigment epithelial cells Disruption of division and migration and atrophy of retina in adult macular degeneration

18

Fibroblasts Disrupted tissue structure locally via secretion of VEGF

1

Melanocytes Reinforcing senescence via secretion of IL-6, IL-8, and PAI-1

19

*Senescence causes different effects in different tissues and cell types.PAI, plasminogen activator inhibitor; VEGF, vascular endothelial growth factor.

SASP

Tumor SuppressionSenescence

IL-6, IL-8,MMPs

Tissue Repair /Tumor Progression

Immune Clearance

IL-1a

Tissue Aging

Self-Sustaining Feedback Loop

Growth Arrest

SenescenceSmuli

Figure 1 The senescence-associated secretoryphenotype (SASP) during aging, cancer develop-ment, and progression. Cellular senescence hasdual effects on human health during the pro-gression of aging and various human disorders.The beneficial effects include tumor suppressionand tissue repair after injury; the deleterious ef-fects include tumor and aging promotion thatinvolve several key SASP molecules, includingIL-6, IL-8, matrix metalloproteinases (MMPs), andspecific miRNAs.

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premature senescence, including chromatin instability, DNAdamage, dysfunctional telomeres, oncogenic mutations,overexpression of cell cycle inhibitors, and stress signals(Figure 2½F2"½F2" ). Oncogenic-induced senescence has beencommonly characterized in vitro using mutant H-RasV12, anoncogene, to induce cell cycle arrest signals by activatingp53 and p16INK4A/pRB pathways.25 p53 contributes tocellular senescence by transactivating genes that blockcellular proliferation, including the p21/Cip1/WAF1 cyclin-dependent kinase inhibitor and miR-34 class ofQ7 miRNAs.26

Similarly, loss of tumor suppressors (PTEN and NF1Q8 ) hasbeen shown to induce premature senescent pathways.27,28

Although our understanding of DNA damageerelatedsenescence provides new insights into these molecularprocesses, stress-induced senescence is currently more afocal point for research on the pathobiology of chol-angiopathies. In vitro, stress-induced senescence has beenmodeled with alterations in culture medium, growth factors,and oxidative stress in various human cell lines.29e32 Inmouse cholangiocytes, proinflammatory cytokines, such astumor necrosis factor a, interferons b and g, have been usedto generate reactive oxygen species and were shown toinduce senescence via the p53/p21WAF1/Cip1 pathway.33 Theevidence linking autoinflammatory cholangiopathies to thispathway will be discussed later.

Potential Mechanisms of Senescence-RelatedHuman Diseases

Senescence-Associated Secretory Phenotype

Senescent cells exhibit altered gene expression and promi-nently secrete cytokines (eg, IL-1Q9 and IL-6), chemokines

(eg, IL-8, CXCL-1, and CXCL-2), insulin-like growthfactorebinding proteins (IGFBPs), and several other factors,including matrix metalloproteinases, serine proteases, andproinflammatory colony-stimulating factors. These solublesignaling factors are referred to as the SASP.34 Acting viaparacrine and autocrine mechanisms, these proteins alter themicroenvironment of the cells, which may lead to adisruption of tissue structure and function (Figure 3 ½F3"½F3").Although SASP has attracted interest for its potentialpathological role in many human diseases, the biologicalfunction of SASP as a regulatory mechanism in tumorsuppression and other areas remains incompletely under-stood. Evidence has emerged related to the antifibroticeffects of SASP in the setting of acute liver injury. Ashepatic stellate cells proliferate and secrete extracellularmatrix (ECM) components and undergo senescence todevelop SASP (Figure 4 ½F4"½F4"), they secrete metalloproteinasesand other proinflammatory molecules that digest the ECMproteins counterbalancing the initial fibrotic response.35

Because senescence has also been reported in chronic bileduct inflammation in the setting of primary biliary cirrhosisand primary sclerosing cholangitis,36 and cholangiocytes inPSC liver exhibit increased expression of SASP compo-nents,15 the senescence program may also limit the fibro-genic response to cholestatic liver injury through the similarHSC Q10-related mechanisms.

Induction of Senescence in Nearby Cells

The ability of SASP to propagate senescence through pos-itive feedback has been hypothesized to potentially be apathological mechanism in cholangiopathies. To date, theevidence for this effect remains limited. The observation of

Division Competent Cell

Oxida!ve Stress

Senescent Cell

SA-β-GALp16INK4a

p21WAF1/cipl

TelomereDysfunc!on

DNA Damage

Oncogenic Ac!va!on

Figure 2 Overview of senescence. Variousfactors, such as telomeric dysfunction, DNA dam-age, oncogenic activation, and oxidative stress,induce senescence in division competent cells. Onbecoming senescent, the cell and its nucleusbecome enlarged and begin to express p16INK4a,p21WAF1/Cip1, and senescence-associated b-galac-tosidase (SA-b-GAL). Senescent cells present alarge flattened morphology and build up a SA-b-GAL activity that distinguishes them from mostquiescent cells.

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clustered expression of p16INK4a in nevi has led to thehypothesis that senescent cells secrete senescence-inducingagents that act on other cells locally.37 Subsequently, inhuman melanoma cell lines, high levels of IGFBP P-rP1(IGFBP-rP1, previously known as IGFBP7 and mac25) inhibitBRAF-MEK-ERKQ11 oncogene signaling and activate senes-cence or apoptotic pathways.38 As discussed later, over-expression of IGFBP-rP1 has also been observed in BA.However, its exact function in the setting of the diseaseremains unclear. IGFBP-5 has also been implicated as a pro-tein, which may act locally to induce senescence. Humanumbilical endothelial cells treated with exogenous rh-IGFBP-5Q12

were found to increase expression of p53 and p21 anddecrease proliferation compared to untreated controls.39

Depletion of Progenitor Cell Populations

The potential pathological link between senescence and thedepletion of progenitor cells is perhaps theoretically moreobvious. The antiproliferative properties of senescence canbe detrimental to essential functions of progenitor cell pop-ulations, which are crucial to tissue repair and regeneration innumerous organs, particularly the liver. It is possible that asinjury or other stimuli deplete these populations, it interfereswith proper tissue function. Some of the most direct evidencefor this pathological mechanism comes from research oncardiac anthracycline toxicity using a rat model. Treatment ofmice with doxorubicin led to expansion of a p16INK4a-positive cardiac stem cell population exhibiting irreversiblegrowth arrest, and this population was significantly associ-ated with left ventricular dysfunction. By using intra-myocardial injections of syngeneic cardiac stem cells,researchers were able to rescue the cardiac function of the

treated animals, which led to significantly improved survivalcompared to untreated controls.40 The functional role of astrong cellular senescence inducer, the homeobox transcrip-tion factor prospero homeobox protein 1 (Prox1), has beenrecently clarified in liver progenitors.41 Prox1 depletion inbipotent hepatoblasts significantly decreased the expressionof multiple hepatocyte genes and led to defective hepatocytemorphogenesis. Consequently, abnormal epithelial structuresexpressing hepatocyte and cholangiocyte markers or resem-bling ectopic bile ducts developed in the Prox1-deficient liverparenchyma. Nevertheless, excessive commitment of hep-atoblasts into cholangiocytes, premature intrahepatic bileduct morphogenesis, and biliary hyperplasia occurred inperiportal areas of Prox1-deficient livers.41 Certainly, thecrucial role of liver progenitor cells could suggest that theremay be some contribution of this model in hepatobiliarydisease as well.

Cholangiopathies

Biliary Atresia

BA is a progressive, fibro-obliterative disease of the extra-hepatic biliary tree that represents the most common causeof pediatric liver transplantation. Even with early Kasaihepatoportoenterostomy, liver transplantation remainsnecessary for 60% to 80% of patients, and without trans-plant, the 5-year survival is an unfortunate 60%.42e44 Thepathophysiology of this disease remains unknown, althougha combination of viral, toxic, genetic, and immunologicaletiologies have been generally considered.Interestingly, senescence markers have been observed in

affected pediatric patients. In two patients with cirrhosis

DNA Damage Foci(CDNA-SCARS/TIF)

Heterochroma!n Foci(SAHF)

-

Growth

SASP

Arrest

p16INK4a

Proinflammatory State

SenescenceSmuli

Nonsenescent Cells(Cholangiocytes)

SA-β-GAL

Figure 3 Formation of senescence-associatedsecretory phenotype (SASP). Senescent cells/cholangiocytes display different phenotypes fromquiescent or terminally differentiated cells (nondi-viding), whereas the definite feature of the senescentphenotype is notably defined. The significant markersof senescent cells/cholangiocytes include an essen-tially irreversible growth arrest; expression ofsenescence-associated b-galactosidase (SA-b-GAL)and p16INK4a; nuclear foci containing DNA damageresponse proteins (DNA-SCARS/TIF Q20) or senescence-associated heterochromatin foci (SAHF); and robustsecretion of various growth factors, cytokines(proinflammatory), proteases, and other proteins,such as SASP. Proinflammatory SASP mediators(C-X-C chemokine receptor 2, IL-6, and IL-6 receptor)can reinforce cellular senescence in an autocrine orparacrine manner. The secretion of SASP is the mostsignificant of these effects because it turns senescentcells/cholangiocytes into a proinflammatory statethat has the ability to promote disease progression.

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secondary to BA, p53 and SA-b-GAL were seen expressed innodular hepatocytes, canals of Hering, and cholangioles withan absent expression of p16 or p21.45 These observationscorrelate with the results of a genome-wide gene expressionanalysis of normal, diseased control, and end-stage BA livers,which found strong expression of IGFBP-rP1 compared to thenormal and non-BA diseased controls. Previously, IGFBP-rP1has been shown to be up-regulated in various cell lines withsenescent phenotypes and has shown to be a p53-responsivegene.46,47 Although the data are limited, taken together,these findings may suggest a possible mechanism for senes-cence of cholangiocytes in BA.

Although previously there have been little data involvingtelomere dysfunction as a mechanism leading to BA, a studyby Sanada et al48 measured hepatocyte telomeres usingQ-FISH and showed preserved telomere length, but normal-ized telomere/centromere ratios, being markedly reducedcompared to healthy controls. Whether these findings repre-sent a true cause or consequence of the BA remains unclear.Previously, estimations of hepatocyte telomere lengths usingQ-FISH in patients with cirrhosis from chronic viral hepatitis,autoimmune hepatitis, primary sclerosing cholangitis, primarybiliary cirrhosis, and alcoholic liver disease have found thattelomere length correlates to Child-Pugh score independent ofassociated disease.49 Although the role of telomere dysfunc-tion in BA remains unclear, these studies suggest that hepa-tocyte telomere length may have some potential role as anadjunct biomarker to clinical scoring models of cirrhosis, suchas pediatric end-stage liver disease, model for end-stage liverdisease, and Child-Pugh in certain diseases.

Primary Sclerosing Cholangitis

PSC is an idiopathic, autoinflammatory disorder characterizedby fibrosis, stricturing, and obliteration of medium and largeducts throughout the biliary epithelium. This incurable diseasehas a prominent associationwith inflammatory bowel disease in70% of patients, particularly ulcerative colitis, and its

progressive nature leads to complications, including cholestasis,hepatic failure, and cholangiocarcinoma.50 Median survival inthe absence of liver transplant is between 10 and 12 years.51,52

The role of cell senescence in PSC remains an emergingfield of research. Cholangiocytes in PSC have been shown tohave increased expression of SA-b-GAL, p16INK4a, andp21WAF1.53 We have demonstrated that two cellular senes-cence markers, plasminogen activator inhibitor-1 and p57,are significantly increased in isolated cholangiocytes fromMDR2 knockout mice, which develop periportal fibrosissimilar to human PSC (unpublished data). Plasminogenactivator inhibitor-1 is an essential mediator of replicativesenescence in vitro and is one of the biochemical fingerprintsof senescence in vivo.54 The p57 overexpression inducedcellular senescence through cell cycle arrest in the G1

phase.55 It has been demonstrated that p57 is involved inhepatocyte growth arrest at two distinct points during liverdevelopment: the perinatal period and the postnatal transitionto a quiescent adult hepatocyte phenotype.56 Recently,Tabibian et al15 Q13demonstrated increased expression ofg-H2Ax, a marker of DNA damage and up-regulation ofSASP markers IL-6, IL-8, chemokine ligand 2, and plas-minogen activator inhibitor-1 in diseased samples. Corollaryto these findings, by using the lipopolysaccharide inflam-matory stress model, they reproduced cholangiocytesenescence-inducing expression of SASP markers andbystander cholangiocyte senescence. Furthermore, theirstudy showed significantly increased expression of N-Rasprotein colocalization with activated RAS in PSC, which wasabsent in PBC, hepatitis C, or control samples. The data buildon their previous work suggesting that N-Ras protein medi-ates lipopolysaccharide-induced inflammation, further sup-porting a potential role for N-Ras signaling in thepathogenesis of PSC, adding a molecular mechanism to ourunderstanding of PSC as an inflammatory disease.57

One recent study has characterized and identifiedphenotypic and signaling features of isolated PSC patient-derived cholangiocytes.58 The cholangiocytes from stage

Figure 4 Recovery effect of hepatic stellatecells drives the senescence-associated secretoryphenotype (SASP) during liver fibrosis. Thesecretion of SASP by activated stellate cells canprevent further proliferation of extracellular ma-trix (ECM)-producing cells, promote ECM degra-dation, and accelerate clearance of activatedhepatic stellate cells from the site of liver injury/fibrosis. COL1A1, type I collagen; COL1A2,collagen 1 chain type II; CTGF, connective tissuegrowth factor; MMP, matrix Q21metalloproteinases;PDF, ---; ROS, reactive oxygen species; TGF,transforming growth factor; TIMP, tissue inhibitorof metalloproteinases.

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4 PSC patient liver explants were isolated by dissection,differential filtration, and immune-magnetic bead separa-tion. The proportion of cholangiocytes staining positivefor senescence-associated b-galactosidase was found to bemuch higher in PSC cholangiocytes compared withcultured normal human cholangiocytes (48% versus 5%;P < 0.01).58 Interestingly, by using Q-FISH, telomerelength in the cholangiocytes of nine PSC samples wasfound to be maintained compared to normal controls.15

Adjacent hepatocyte telomere length was not reported.If the data from Wiemann et al49 on shortened hepatocytetelomeres in PSCs are substantiated, a complex interac-tion of senescent pathways could be involved in thepathogenesis of this disease. Certainly, differences instages of liver disease may play a role, but further clari-fication of the data on the telomere length of hepatocytesand cholangiocytes in PSCs may be helpful.

Cellular Rejection

Acute CR has historically been associated with a progressive,obliterative cholangiopathy. Histopathology shows a non-suppurative cholangitis of the interlobular bile ducts withcholangiocytes exhibiting cellular and nuclear enlargement,multinucleation with uneven nuclear spacing, and thickeningof the basement membrane.59 In recent years with moreaggressive immunosuppression, the rate of CR has decreasedfrom 60% to 70% to 15% to 20% in graft recipients, withductopenia becoming even rarer.60 Despite therapeutic ad-vances, CR remains a significant cause of morbidity and costin liver transplant.

Even in early stages of CR, senescence has beenobserved with absent proliferation of cholangiocytesdespite their stereotypical proliferative response toinjury.61 The number of p21WAF1/Cip1-positive chol-angiocytes has been shown to correlate with the Banffgrade of acute rejection and declines with treatment.62,63

Previously, it has been observed that patients with failedallografts who had received cyclosporine had more duc-topenia than patients who had received tacrolimus.64

Interestingly in other epithelial tissues, cyclosporine,but not tacrolimus, has been shown to augment trans-forming growth factor-b expression, which is known toinduce p21WAF1/Cip1 expression.65 Building on thesefindings, Brain et al14 presented observational data onS100A4 expression in CR and in an in vitro model thatdescribed dedifferentiation driven by transforming growthfactor-b2. Given the diverse expression of S100A4, theprecise implications of this work are uncertain, yetintriguing.

Primary Biliary Cirrhosis

Primary biliary cirrhosisQ14 (PBC) is a progressive, autoim-mune cholangiopathy involving the small intrahepatic bileducts. The disease tends to affect women >40 years who

may present with symptoms of cholestasis, fibrotic biliarylesions, and hepatomegaly.66,67 Over time if untreated, thesustained loss of bile duct epithelial cells in PBC leads tocirrhosis and liver failure. Serum antimitochondrial anti-bodies can be detected in the 90% of cases. These anti-bodies target the E2 and E3BP subunits of the pyruvatedehydrogenase complex (PDC-E2 and PDC-E3BP).68,69

However, their involvement in the pathogenesis of PBChas been uncertain.Sasaki et al53 Q15have led research on the role of cellular

senescence in pathogenesis of PBC. The authors haveshown that PBC cholangiocytes exhibit senescence markersSA-b-GAL, p16INK4, and p21WAF1/Cip far more frequentlythan normal and viral hepatitis controls and observed theincreased presence of infiltrating myeloperoxidase-positiveinflammatory cells in PBC.53 The role of the inflammatorycells remains unclear, but it suggests that oxidativestresseinduced senescence may contribute to the patho-genesis of PBC. Examining telomere length using Q-FISH,they demonstrated that diseased small bile ducts and duct-ules in PBC compared with normal-appearing bile ducts inPBC, chronic viral hepatitis, and normal livers had signifi-cantly shorter telomeres, although the role of telomerelength in PBC is unknown.More interestingly, autophagy, the lysosomal pathway

involving the catabolism of cellular components, has beenidentified as a necessary component to the activation of cellsenescence.70 Sasaki et al Q16showed that senescent PBCcholangiocytes accumulated markers of autophagy,including microtubule-associated proteins-light chain 3b,cathepsin D, and lysosome-associated membrane protein-1.They further showed aggregation of p62 sequestosome-1, aspecific marker for autophagy, in diseased small bile ducts,suggesting some impairment of autophagy in PBC.2,70e72

Subsequently, the group has shown colocalization of mito-chondrial antigen PDC-E2 with autophagy marker lightchain 3b and increased in vitro cell surface expression ofPDC-E2 in cultured BECs Q17on exposure to various stresses.73

Taken together, these findings provide insight into therelationship between autophagy, the expression of mito-chondrial antigens, and autoimmunity in PBC. The exactmechanisms behind this pathological interaction remainunclear, but certainly this work suggests further under-standing of the pathways related to senescence, particularlyautophagy, may yield new insights in the development ofbiliary disease.

Conclusion and Perspectives

Cellular senescence is linked to the development of varioushuman diseases, and its role in biliary disease remains to becompletely elucidated. Even as recent data suggest newquestions related to the mechanisms of senescence in thedevelopment of cholangiopathies, the role cellular senes-cence plays in the regulation of biliary growth, injury, and

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response to injury continues to expand our understanding ofbiliary disease and hopefully, in the future, yield new op-portunities for targeted therapies.

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