Liver Renewal: Detecting Misrepair and Optimizing Regeneration

SYMPOSIUM ON REGENERATIVE MEDICINE

Liver Renewal: Detecting Misrepair and Optimizing Regeneration Mariana Verdelho Machado, MD, and Anna Mae Diehl, MD CME Activity From the Division of Gastroenterology, Duke University, Durham, NC.

Target Audience: The target audience for Mayo Clinic Proceedings is primarily internal medicine physicians and other clinicians who wish to advance their current knowledge of clinical medicine and who wish to stay abreast of advances in medical research. Statement of Need: General internists and primary care physicians must maintain an extensive knowledge base on a wide variety of topics covering all body systems as well as common and uncommon disorders. Mayo Clinic Proceedings aims to leverage the expertise of its authors to help physicians understand best practices in diagnosis and management of conditions encountered in the clinical setting. Accreditation: Mayo Clinic College of Medicine is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. Credit Statement: Mayo Clinic College of Medicine designates this journalbased CME activity for a maximum of 1.0 AMA PRA Category 1 Credit(s).
Physicians should claim only the credit commensurate with the extent of their participation in the activity. Learning Objectives: On completion of this article, you should be able to (1) identify the major cell types involved in fibrogenesis and liver regeneration, (2) identify major pathways implicated in activation of hepatic stellate cells into myofibroblasts, (3) recognize the intricate relationship between progenitors and hepatic stellate cells, and (4) criticize the difficulty of interpreting cell tracing studies, acknowledging the plasticity of cells that change expression of typical markers across time, in an injured liver. Disclosures: As a provider accredited by ACCME, Mayo Clinic College of Medicine (Mayo School of Continuous Professional Development) must ensure balance, independence, objectivity, and scientific rigor in its educational activities. Course Director(s), Planning Committee members, Faculty,

and all others who are in a position to control the content of this educational activity are required to disclose all relevant financial relationships with any commercial interest related to the subject matter of the educational activity. Safeguards against commercial bias have been put in place. Faculty also will disclose any off-label and/or investigational use of pharmaceuticals or instruments discussed in their presentation. Disclosure of this information will be published in course materials so that those participants in the activity may formulate their own judgments regarding the presentation. In their editorial and administrative roles, William L. Lanier, Jr, MD, Terry L. Jopke, Kimberly D. Sankey, and Nicki M. Smith, MPA, have control of the content of this program but have no relevant financial relationship(s) with industry. The authors report no competing interests. Method of Participation: In order to claim credit, participants must complete the following: 1. Read the activity. 2. Complete the online CME Test and Evaluation. Participants must achieve a score of 80% on the CME Test. One retake is allowed. Participants should locate the link to the activity desired at http://bit.ly/ 1ifgvUo, On successful completion of the online test and evaluation, you can instantly download and print your certificate of credit. Estimated Time: The estimated time to complete each article is approximately 1 hour. Hardware/Software: PC or MAC with Internet access. Date of Release: 01/01/2014 Expiration Date: 12/31/2015 (Credit can no longer be offered after it has passed the expiration date.) Privacy Policy: http://www.mayoclinic.org/global/privacy.html Questions? Contact [email protected].

Abstract Cirrhosis and liver cancer, the main causes of liver-related morbidity and mortality, result from defective repair of liver injury. This article summarizes rapidly evolving knowledge about liver myofibroblasts and progenitors, the 2 key cell types that interact to orchestrate effective repair, because deregulation of these cells is likely to be central to the pathogenesis of both cirrhosis and liver cancer. We focus on cirrhosis pathogenesis because cirrhosis is the main risk factor for primary liver cancer. Emerging evidence suggests that the defective repair process has certain characteristics that might be exploited for biomarker development. Recent findings in preclinical models also indicate that the newly identified cellular and molecular targets are amenable to therapeutic manipulation. Thus, recent advances in our understanding about key cell types and fundamental mechanisms that regulate liver regeneration have opened new avenues to improve the outcomes of liver injury. Trial Registration: clinicaltrials.gov Identifier: NCT01899859 ª 2014 Mayo Foundation for Medical Education and Research

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he tremendous regenerative capabilities of the liver have been suggested since the ancient Greek myth of Prometheus. Today, we know that the myth closely resembles actual physiology because the liver can fully regenerate after removal of 70% of its mass, even after repeated hepatectomies.1 However, it is equally true that regeneration 120

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sometimes fails, resulting in misrepair of liver injury and consequent progressive scarring (cirrhosis) and/or development of liver cancer. In this article, we focus on misrepair that results in liver cirrhosis, the main risk factor for primary liver cancer,2,3 because recent reviews have been written about the pathogenesis of primary liver cancer and the association

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between deregulated regeneration and hepatocarcinogenesis.4,5 At present, therapies for most types of chronic liver disease focus on eliminating the causes of liver injury while relying on inherent hepatic repair capabilities to restore liver structure and function. When these approaches fail, either the diseased liver is replaced by transplant or complications resulting from liver damage are palliated until death ensues. The burden of liver damage is increasing worldwide owing to recent epidemics of viral hepatitis6 and obesity-related fatty liver disease7 that have been superimposed on endemic liver diseases caused by alcohol, other toxins, and relatively rare inherited conditions. Even in countries with robust resources fordand social acceptance ofdorgan transplant, the demand for liver replacement now far outstrips the availability of transplantable organs.8 This situation is compounded by the social and economic forces that stifle organ donation and transplant. Thus, health care delivery systems are confronted by a growing demand for palliative liver care, which is expensive and only somewhat successful in reducing liver-related morbidity but incapable of preventing ultimate mortality from advanced liver damage. Thus, there is a definite need to develop effective therapeutic approaches to improve repair and regeneration of injured livers. Another key challenge is to improve the detection of individuals with defective liver repair before overt, life-threatening manifestations of liver damage emerge. This detection is important so that pro-regenerative treatments can be implemented when they have the greatest chance of efficacy. Success in both diagnosis and treatment necessitates improved understanding of the mechanisms that regulate effective liver regeneration. SCIENTIFIC OVERVIEW Liver Regeneration Is Nothing More (or Less) Than a Wound-Healing Response Liver regeneration is nothing more (or less) than a wound-healing response. As such, it is a precisely orchestrated, multifaceted process that involves inflammation, vasculogenesis, matrix remodeling, and the growth and differentiation of liver progenitors. It has long been believed that the wounded liver is superior to other adult Mayo Clin Proc. n January 2014;89(1):120-130 www.mayoclinicproceedings.org

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organs in regenerating its functional epithelia mainly because surviving mature hepatocytes are able to reenter the cell cycle and replicate repeatedly.9 Until recently, this dogma substantially curtailed research about liver progenitors. Thus, relatively little is known about these cells in adults. However, knowledge in this area is improving owing to growing evidence that stem/progenitor cells are generally required for effective wound-healing responses in liver, as in other organs.10 Indeed, the progenitor compartment appears to be the main source for the replacement of dead liver epithelial cells during most types of chronic liver disease because chronic exposure to injury-related stresses typically triggers replicative senescence in mature liver cells that are able to survive.11 That said, it is not known why mammalian liver is much more effective than other vital organs at regenerating its parenchyma because many aspects of wound healing are highly conserved in organs and across species. Solving this puzzle might suggest new interventions to optimize regeneration not only of wounded livers but also of other organs. Effective Wound Healing Involves Constrained Scarring One of the most highly conserved components of the wound-healing response is scarring. Scarring describes a tissue remodeling response that is typically triggered by “deep” injury. It involves accumulation of cell types that are fairly inconspicuous in the healthy tissue, including myofibroblasts (MFs), various immune system cells, activated endothelial cells, and progenitors. These cells interact to initiate the reconstruction of the injured tissue via a process that involves removing cellular debris, remodeling existing stroma vasculature, and marshaling the growth and differentiation of cells that will ultimately replace damaged parenchyma while attempting to maintain the viability of surviving parenchyma until fully functional replacement cells become available. In its entirety, scarring is an essential component of effective regeneration. However, it is often maligned because tissue architecture becomes variably disrupted by the accumulation of “abnormal” matrix during much of this rebuilding response, and remodeling the matrix back to its “healthy” configuration is easily thwarted if injury recurs. Thus, fibrosis is often considered to be synonymous with scarring. In

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reality, however, fibrosis is merely one stage of that multifaceted wound-healing response. Deregulated Scarring Inhibits Regeneration and Causes Misrepair of Injury Because progressive fibrosis is a hallmark of deregulated scarring (and resultant “misrepair”), attention has been focused on characterizing the cell types that produce fibrotic matrix and mechanisms that control that process. Four potential sources of fibrogenic liver cells have been identified: MFs derived from resident vascular pericytes (also known as hepatic stellate cells [HSCs]) or portal fibroblasts, fibrocytes derived from infiltrating bone marrowederived mononuclear cells, and resident liver epithelial cells that undergo an epithelial-to-mesenchymal transition (EMT).12 Current opinion favors the concept that MFs are mainly responsible for liver fibrosis and suggests that the cellular source of these MFs differs according to the location and duration of liver injury. Portal fibroblasts are the key source of MFs during early biliary injury,13 whereas MFs are derived mainly from HSCs during acute and chronic hepatic parenchymal injury,14,15 including that which occurs as biliary injury progresses.16 PRECLINICAL STUDIES Liver Scarring Involves Accumulation of MFs Derived From HSCs The pivotal role of HSCs in liver fibrosis was confirmed recently by studying transgenic mice, in which regulatory elements of the glial fibrillary acidic protein gene (GFAP) were used to direct stellate celleselective killing by ganciclovir. The results proved that selectively deleting HSCs during liver injury abolished liver fibrosis,17 thus emphasizing the importance of understanding the mechanisms that control the accumulation of HSC-derived MFs during recovery from liver injury. The process by which MFs are derived from HSCs can be modeled by culturing freshly isolated primary HSCs in serum-containing medium on plastic dishes. Thus, this in vitro system has been extensively used to delineate the mechanisms involved in HSC “transdifferentiation.”18,19 However, studies comparing the expression profiles of MFs generated in vitro and in vivo have identified some differences, suggesting that the in vitro system might not perfectly recapitulate all the cues that modulate 122

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HSC transdifferentiation in injured livers.20,21 Nevertheless, factors that promote the in vivo activation of nonproliferative, nonfibrogenic (ie, quiescent) HSCs into proliferative, fibrogenic HSC-derived MFs are generally effective in vitro, and hence, the in vitro system has been widely used to characterize the signaling mechanisms involved in the formation of MF from quiescent HSCs. Transforming growth factor b (TGF-b), a major profibrogenic factor in many tissues, is a key factor that drives the formation of MFs from HSCs22; however, various other factors are also involved, including other cytokines (eg, connective tissue growth factors23 and interleukin 1324,25), hormones (eg, leptin26,27), vasoactive factors (eg, angiotensin28), morphogens (eg, hedgehog19 and notch ligands29), and the stiffness of the matrix itself.30 Virtually all these fibrogenic factors (and many others) are induced at one point or another during the woundhealing process. Thus, it is likely that they all interact to control MF accumulation, and thus fibrogenesis, in vivo. However, at present, it remains unclear to what extent the factors regulate unique, redundant, or conserved mechanisms that control HSC fate, despite the fact that downstream signaling pathways have been delineated for each of these individually. Also obscure is whether or not liver injury imposes some sort of hierarchy on the relative importance of various factors for regulating HSC/MF behavior in vivo. These issues have been difficult to address by studying rodent strains with either naturally occurring or genetically engineered constitutive deletions of individual factors because the very fact that the animals survive to adulthood suggests that compensatory mechanisms have been evoked to adapt to chronic inactivation of any one factor that regulates MF accumulation during liver growth and regeneration. Recent advances that permit conditional deletion of key signaling molecules in a cellselective fashion have overcome some of the aforementioned challenges, providing new, biologically relevant insights into fundamental mechanisms that control MF accumulation during liver injury. Surprisingly, the conditional deletion of a single molecule, smoothened (Smo), in cells expressing alpha smooth muscle actin (aSMA, a widely accepted marker of MFs), including HSCs, virtually abolished the accumulation of MFs in several different murine

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liver injury models.31 Smoothened is a transmembrane protein that is best known for its role in propagating signals that result in the stabilization and nuclear accumulation of glioblastoma (Gli) family transcription factors. Smoothened activity is tonically suppressed by another transmembrane protein, patched (Ptc), when Ptc is not interacting with its ligands, sonic hedgehog, Indian hedgehog, or desert hedgehog. However, binding of hedgehog (Hh) ligands to Ptc relieves repression of Smo, permitting signaling, which ultimately culminates in the nuclear accumulation of Gli factors with resultant effects on the transcription of Gli-regulated genes.32 Gli target genes include factors that inhibit apoptosis (eg, Mcl-1),33 control liver progenitor fate (eg, Sox9),34 and promote liver fibrosis (eg, osteopontin),35 helping us to explain some of the other unanticipated consequences that followed Smo deletion in MFs. Smoothened also interfaces with other signaling pathways. For example, it has long been known to modulate signaling initiated by TGF-b36 and G proteine coupled receptors,37 and it was recently found to gate signals downstream of leptin and notch receptors.27,29 Thus, Smo appears to represent a key “node” that integrates signals initiated by an array of cell surface receptors that respond to diverse factors that regulate various aspects of MF biology. This permits MFs to modify their phenotype according to dynamic cues emanating from the changing microenvironment within injured livers (Figure). Deriving MFs From HSCs Requires an Epithelial to MesenchymaleLike Process and Is Reversible As mentioned earlier, most of the MFs that accumulate during various types of liver injury derive from resident HSCs. An analysis of freshly isolated and culture-activated primary HSCs reveals that HSCs are Hh-responsive cells and that HSCs begin to produce Hh ligands and up-regulate Hh signaling as they transdifferentiate to become MFs.38,39 Moreover, Hh signaling is required for HSCs to remain MFs because knocking down Smo (or inhibiting Hh ligand activity with neutralizing antibodies) causes the HSC-derived MFs to transition back to a more quiescent (ie, nonfibrogenic, nonproliferative, nonmigratory) phenotype, both in vitro and in vivo.19,31 Thus, canonical Hh signaling (ie, Hh ligand/ Mayo Clin Proc. n January 2014;89(1):120-130 www.mayoclinicproceedings.org

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receptor initiated signaling via Smo) promotes the transition of HSC into a myofibroblastic phenotype, whereas blocking Hh signaling facilitates their transition out of the myofibroblastic state. Although the concept that HSCs are capable of such transitions was initially challenged, our group and at least 2 other independent laboratories recently confirmed that HSC-derived MFs are able to transition back to a nonmyofibroblastic phenotype in situ.40,41 More surprisingly, sequential immunohistochemistry establishes a strong reciprocal relationship between Hh pathway activity and HSC expression of E-cadherin.31,42 This finding has been confirmed

FIGURE. In the normal liver, residual hepatocytes can replicate to regenerate hepatocytes that die. However, in chronic liver diseases, cytotoxic stresses cause hepatocytes to acquire a phenotype of replicative senescence. The injured and dying hepatocytes also send signals to neighboring progenitor cells and hepatic stellate cells to promote a wound-healing response. For example, wounded hepatocytes release morphogens that are known to promote fibrogenesis, such as hedgehog and Wnt ligands. In response to these factors, the hepatic stellate cell compartment becomes myofibroblastic and expands. Progenitor growth is also increased, inflammatory cells are recruited, and vasculogenesis is induced. The accumulating myofibroblasts, progenitors, and neovessels tend to intermingle within and along fibrous matrix, forming scar tissue. Scar formation occurs transiently during successful regeneration. However, when fibrogenic mechanisms are sustained or excessive, scarring becomes progressive and regeneration of functional hepatic parenchyma is compromised. Instead, the deregulated remodeling response promotes cirrhosis. The cirrhotic microenvironment, in turn, favors the outgrowth of malignant liver epithelial cells, thereby promoting hepatocarcinogenesis.

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by the quantitative real-time polymerase chain reaction analysis of isolated HSCs and whole liver RNA during periods of low and high Hh pathway activity. E-cadherin is a component of junctional complexes, and its expression is a characteristic of epithelial cells.43 In healthy livers, junctional complexes between HSCs and hepatocytes have long been revealed by electron microscopy.44 The aggregate data, therefore, suggest that HSCs are normally part of the hepatic epithelia but undergo an epithelial to mesenchymalelike transition that permits them to acquire a more myofibroblastic phenotype during liver injury. This discovery, in turn, helps us explain why TGF-b, a known EMT-promoting factor,45 is important for the myofibroblastic transdifferentiation of HSCs,46-48 as well as several earlier reports that indicated that the overexpression of bone morphogenic protein-7 (BMP-7), a potent EMT inhibitor, prevented and/or reversed fibrosis in various rodent liver injury models.49-51 MFs and Liver Progenitors: More Than Intimate Neighbors During Scarring? Like MFs, cells expressing progenitor markers are fairly inconspicuous in healthy adult livers, but rapidly accumulate during various types of liver injury.52-55 Interestingly, progenitors and MFs generally intermingle in areas of scarring. In portal areas, progenitors often cluster to form duct-like structures embedded within a fibrotic stroma that is enriched with MFs, inflammatory cells, and neovessels. This process has been dubbed the “ductular reaction,” and it sometimes extends finger-like projections into the hepatic parenchyma, bridging adjacent portal tracts with fibrous septae.56 The portal basis for the ductular reaction is consistent with the concept that adult liver progenitors reside within the biliary tree. The precise localization of biliary-associated progenitors remains a topic of hot debate, however. Although it has long been believed that the most proximal branches of the biliary system (ie, canals of Hering) harbor bipotent progenitors that are capable of generating either hepatocytes or cholangiocytes,57,58 recent evidence suggests that a more primitive multipotent progenitor compartment may reside within the submucosal glands within the walls of larger bile ducts.59 These multipotent progenitors may be able to generate 124

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pancreatic cells as well as liver cells.59 Emerging evidence suggests that HSCs can also generate liver epithelial cells in injured livers.31,60-62 This concept is consistent with the fact that the space of Disse exhibits many features of a classical progenitor niche63 as well as evidence that pluripotent progenitors are members of pericyte populations in certain other tissues.64-67 Pluripotent progenitors are stem-like cells, capable of generating progeny of different lineages. Studies of induced pluripotent stem cells indicate that the process of global reprogramming requires epithelial to mesenchymale and mesenchymal to epithelialelike transitions.68-70 In this regard, it is notable that HSCs are the only resident liver cell type that has been proven to undergo epithelial to mesenchymale and mesenchymal to epithelialelike transitions in situ.31 The possibility that liver injury might induce adult HSCs (or other resident liver cells) to become multipotent progenitor cells is particularly provocative because Espejel et al71 reported that the adult hepatocytic progeny of mouse embryonic fibroblastederived induced pluripotent stem cells can acquire the functional and proliferative capabilities of normal mature hepatocytes.71 More recently, Takebe et al72 generated liver-like structures (dubbed liver buds) by coculturing hepatic endoderm cells from human mouse embryonic fibroblastederived induced pluripotent stem cells with endothelial and mesenchymal lineages. When transplanted into mice, the liver buds rapidly developed a vascular network that was connected to the host vasculature and acquired the histological organization and functional features that resembled normal liver, including albumin production and drugmetabolizing properties.72 Thus, strong experimental evidence supports the concept that fully functional hepatocytes can be derived from primitive progenitors during adulthood. Much of the lingering difficulties in clarifying the role(s) of endogenous progenitors in adult liver regeneration (and using this knowledge to optimize liver repair) reflects the inherent plasticity of progenitor cells.73 In other words, progenitors are “changeable” and rapidly modify their phenotype to adapt to changes in their microenvironment, which means that cells that express certain markers in situ may stop expressing these markers, but gain expression of other markers, during isolation and/or culture. Another

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challenge is imposed by the fact that progenitors give rise to progeny. Hence, the differentiation process itself invariably “contaminates” initially pure populations of progenitors. Because progeny generally retain some of the markers of their parents for at least a while, it is risky to rely on a single marker to identify a cell as being a progenitor, making more complex characterization necessary. However, demonstrating coexpression of multiple markers within a single cell has proven to be technically challenging in situ and progenitor plasticity inherently jeopardizes the validity of in vitro phenotyping. Thus, at present, we know little about which endogenous progenitor populations are mobilized, or how this process is regulated, during recovery from liver injury. To overcome the aforementioned obstacles, investigators have carefully characterized the hepatic specification of induced pluripotent stem cells74-76 and relied on functional assays, such as serial cell transplant and organoid formation,77 to determine whether a particular cell type has progenitor capabilities. By using these approaches, cells that express certain surface proteins (eg, CD24, CD133, CD26, CXCR4, LGR5, EpCAM, and Smo) have been identified as adult liver progenitors on the basis of their ability to generate both hepatocyte-like and cholangiocytic cells.59,77-80 Notably, some of the liver progenitoreassociated cell surface proteins interact with soluble factors that are known to regulate the fate of multipotent progenitors in other tissues. For example, CXCR4 is the receptor for stromal-derived factor 1, a growth factor for hematopoietic progenitors81; Lgr5 interacts with R-spondins to enhance Wnt signaling in intestinal progenitors82,83; Smo propagates Hh ligandeinitiated signals that regulate fate decisions in skin and neural progenitors.84-86 Moreover, different types of cells that accumulate in injured livers (eg, wounded hepatocytes, inflammatory cells, activated macrophages, immature liver cells, and MFs) produce one or more of these factors.87-90 At present, however, there is limited solid evidence, with “gold standard” lineage tracing studies, that support or refute the utility of progenitor markers in the identification of which progenitors participate in the regenerative responses to adult liver injury. Lineage tracing is done by exploiting cell typeeselective promoter elements to “tattoo” putative progenitors and the progeny that they Mayo Clin Proc. n January 2014;89(1):120-130 www.mayoclinicproceedings.org

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generate, thereby permitting progeny to be “tracked” even after they lose other parental cell markers.91 The information derived from lineage tracing studies during adult liver injury is extremely limited and can be briefly summarized as follows. Hepatocytic cells were recently derived from Lgr5þ progenitors after acute CCL4-induced liver injury.77 Some ductular appearing cells and a few periportal hepatocytes were derived from cells expressing the transcription factor FoxL1.92 Two reports indicated that hepatocytes, cholangiocytes, and stellate cells were derived from cells that expressed either GFAP or aSMA after bile duct ligation or liver damage induced by feeding methionine- and choline-deficient diets supplemented with ethionine.31,61 Because HSCs are the only type of resident liver cells that are known to express both GFAP and aSMA, and Smo is known to regulate stellate cell fate, the later studies support various other lines of evidence suggesting that Hh-responsive HSCs are multipotent progenitors. For example, several groups have reported that HSCs express various markers of progenitors and stem-like cells, including Nanog, Oct4, CD133, CD26, CD44, and Fn14.19,31,60,93,94 There is also a report that pancreatic stellate cells differentiated into hepatocytes and ductular cells when transplanted after partial hepatectomy.95 The concept that HSCs might be progenitors for liver epithelial cells is further supported by a recent report demonstrating that the conditional deletion of Smo in HSC-derived MFs prevented accumulation of hepatocytic and ductular progenitors (including FoxM1- and Lgr5-expressing cells) in murine models of liver injury,31,42 complementing the aforementioned lineage-tracing evidence that hepatocytes and ductular cells derive from precursors that express GFAP and aSMA.31 In summary, the existing data indicate that (1) adult livers definitely harbor progenitors, (2) these progenitors are mobilized routinely during the scarring phase of liver regeneration, and (3) the progenitor populations are heterogeneous, that is, composed of progenitors with varying levels of plasticity. Hepatocytes and cholangiocytes appear to arise from a common progenitor. Within the past 5 years, a new “twist” to that paradigm has emerged: that the bipotent liver epithelial progenitor may be the HSC, a type of pericyte that is also capable of acquiring myofibroblastic features.

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Alternatively, a yet-to-be-identified common, multipotent progenitor might give rise to stellate cells (and stellate cellederived MFs), cholangiocytes, and hepatocytic cells during adult liver repair. In either case, growing evidence supports the concept that 2 of the key cell types that emerge during liver scarring, that is, HSC-derived MFs and progenitors, are mutually interdependent, relying on each other to orchestrate liver repair. This insight, in turn, has broad diagnostic and therapeutic implications. CLINICAL STUDIES, CHALLENGES, AND OPPORTUNITIES Leveraging Knowledge About Regeneration to Inform Biomarker Development As mentioned during the beginning of this review, a key unmet need in clinical hepatology is the ability to test noninvasively to identify presymptomatic individuals who are unable to constrain scarring and thus destined to develop cirrhosis and/or primary liver cancer. The previous section suggests that circulating biomarkers that reflect activation of pathways that promote progressive generation/accumulation of scarring-associated cells might be useful for diagnosing individuals who are at high risk for cirrhosis or liver cancer. In this regard, it is noteworthy that increased levels of Hh ligands were reported in rats with early biliary fibrosis, suggesting that this might be a marker of subclinical liver scarring.96 However, we could find no report that mentions that serum testing for Hh ligands has been examined as a cirrhosis (or liver cancer) biomarker in humans, despite several reports demonstrating tight correlations between the levels of Hh pathway activity and severity of the ductular reaction and liver fibrosis in patients with various types of liver disease,89,97-100 as well as evidence linking the level of Hh pathway activation with hepatocellular carcinoma outcomes in humans.101-103 In contrast, increased levels of osteopontin (an Hh-regulated factor that promotes both fibrogenesis and progenitor outgrowth) were found in a small series of patients with nonalcoholic fatty liver diseasee related liver fibrosis.35 Further research to examine the utility of blood biomarkers of Hh and other repair pathways as biomarkers of impending cirrhosis/cancer is warranted. 126

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Indeed, a recent microarray analysis of human liver biopsies revealed that the liver gene signature that distinguishes advanced from early liver fibrosis reflects differential induction of various Hh target genes, including osteopontin and Sox9 (an undisputed marker of multipotent liver progenitors).104 At present, however, clinicians rely on combinations of noninvasive tests (eg, blood tests, physical examination, and abdominal imaging modalities) and liver biopsies to track liver repair (and misrepair) in individual patients over time so that treatment recommendations can be adjusted appropriately. Uncharted TerritorydRegulating Progenitors to Optimize Regeneration A considerable clinical “gap” is the present dearth of effective medical treatments to prevent or treat cirrhosis and liver cancer. The earlier discussion predicts that treatments that are able to transition HSC-derived MFs back to a more quiescent (ie, less mesenchymal) state would control scarring and might also constrain the growth of primary liver cancers by blocking the generation/ accumulation of potential tumor initiating/ stem-like cells. There is evidence to support both concepts in animal models. For example, several studies report that different approaches that increase BMP-7, a potent EMT inhibitor, are able to reverse cirrhosis in rodent models of toxin-induced cirrhosis.105,106 Antibodies and aptamers that neutralize osteopontin also inhibit HSCs from becoming or remaining myofibroblastic.35 Like BMP-7, these agents were also reported to prevent cirrhosis in animals.107 Similarly, systemic administration of a direct pharmacologic antagonist of Smo that abrogates canonical Hh signaling and causes HSC-derived MFs to regain a more quiescent phenotype was reported to reverse established liver fibrosis and cause involution of various types of liver cancer in a mouse model of progressive liver fibrosis and hepatocarcinogenesis.108 That same agent was recently reported to inhibit liver fibrosis in a mouse model of diet-induced nonalcoholic steatohepatitis.109 Smoothened antagonists have also been reported to inhibit the growth of primary cholangiocarcinomas in mice.33 At present, drugs that block Hh signaling have been reserved for use as chemotherapeutic agents in nonliver cancers.110,111 However, the aforementioned preclinical liver data suggest that it might be

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worthwhile to examine the safety/efficacy of these and other Food and Drug Administratione approved agents that antagonize Smo for preventing/treating cirrhosis and liver cancer. As discussed below, the relatively disappointing history of past attempts to tackle these disorders suggests that this work will not be trivial. At present, there are at least 2 putative antifibrotic agents that are being examined via multicenter clinical trials: a lysyl oxidase inhibitor and galectin-3. Lysyl oxidase is an extracellular enzyme that promotes stabilization of collagen fibrils and the integrity of elastin. Galectin-3 is a lectin implicated in MF proliferation, fibrogenesis, and tissue repair. An ongoing phase 1 clinical trial (NCT01899859) is evaluating the role and safety profile of intravenous administration of a galectin inhibitor, GR-MD-02, in patients with biopsy-proven NASH with advanced fibrosis. Both studies aim to restore effective liver regeneration by correcting the abnormally fibrogenic microenvironment that perpetuates MF accumulation in individuals with ongoing liver injury. Historical Baggage But a Bright Future The concept of manipulating regeneration to improve the outcome of acute or chronic liver injury in humans is not new, and previous attempts to optimize liver repair have generally failed to report survival benefits. Generally, these interventions were tested in patients with severe acute, or acute-on-chronic, liver failure (eg, liver assist devices, infusions of hepatocyte growth factor, and administration of bone marrowe derived mesenchymal stem cells).112-117 There are several potential explanations for the previous disappointing outcomes, including the possibility that the interventions themselves were ineffective or applied so late in the disease process that the liver damage itself was no longer reversible. Alternatively, the treatments might actually have improved liver repair, but they have been insufficient to correct all the systemic abnormalities that accompany advanced liver failure and thus death ensued despite improved liver regeneration. These insights underscore the importance of thoughtful patient selection and study design when moving forward in this arena. However, there is a strong rationale for optimism, because emerging natural history data from large populations of patients with viral hepatitis provide compelling evidence that established Mayo Clin Proc. n January 2014;89(1):120-130 www.mayoclinicproceedings.org

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cirrhosis often regresses “spontaneously” once triggers that perpetuate scarring responses are removed.118 In contrast, the rising burden of primary liver cancer suggests that bad outcomes of once-defective regeneration may persist long after ostensibly normal liver structure/function has been restored, emphasizing the importance of developing cost-effective strategies to monitor high-risk populations for defective liver repair. CONCLUSION Knowledge about the fundamental mechanisms that control adult liver regeneration is challenging old dogma and establishing a new conceptual framework that has already identified new diagnostic and therapeutic targets. As the means to apply these discoveries are actualized and introduced into clinical practice, society will learn whether the new approaches enable us to improve liver-related mortality, which has been stagnant for the past 50 years despite advances in diagnosis and treatment of many liver diseases. In any case, hepatologists and their patients are on the cusp of a new era of liver disease therapeutic agents that seek to regenerate damaged livers by exploiting the inherent plasticity of adult liver cells. Abbreviations and Acronyms: aSMA = alpha smooth muscle actin; BMP-7 = bone morphogenic protein-7; EMT = epithelial-to-mesenchymal transition; GFAP = glial fibrillary acidic protein; Gli = glioblastoma; Hh = hedgehog; HSC = hepatic stellate cell; MF = myofibroblast; Ptc = patched; Smo = smoothened; TGF-b = transforming growth factor b Correspondence: Address to Anna Mae Diehl, MD, Division of Gastroenterology, Duke University, Snyderman Bldg, Suite 1073, Durham, NC 27710 (annamae.diehl@ dm.duke.edu).Individual reprints of this article and a bound reprint of the entire Symposium on Antimicrobial Therapy will be available for purchase from our website www. mayoclinicproceedings.org.

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