Progenitor cells in liver regeneration: molecular responses controlling their activation and expansion

Authors


  • Invited Review.

Hanne Cathrine Bisgaard, Department of Medical Biochemistry and Genetics, The Panum Institute, Bldg. 6.5.40, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark. e-mail: hcb@imbg.ku.dk

Abstract

Although normally quiescent, the adult mammalian liver possesses a great capacity to regenerate after different types of injuries in order to restore the lost liver mass and ensure maintenance of the multiple liver functions. Major players in the regeneration process are mature residual cells, including hepatocytes, cholangiocytes and stromal cells. However, if the regenerative capacity of mature cells is impaired by liver-damaging agents, hepatic progenitor cells are activated and expand into the liver parenchyma. Upon transit amplification, the progenitor cells may generate new hepatocytes and biliary cells to restore liver homeostasis. In recent years, hepatic progenitor cells have been the subject of increasing interest due to their therapeutic potential in numerous liver diseases as alternative or supportive/complementary tools to liver transplantation. While the first investigations on hepatic progenitor cells have focused on their origin and phenotypic characterization, recent attention has focused on the influence of the hepatic microenvironment on their activation and proliferation. This microenvironment comprises the extracellular matrix, epithelial and non-epithelial resident liver cells, and recruited inflammatory cells as well as the variety of growth-modulating molecules produced and/or harboured by these elements. The cellular and molecular responses to different regenerative stimuli seem to depend on the injury inflicted and consequently on the molecular microenvironment created in the liver by a certain insult. This review will focus on molecular responses controlling activation and expansion of the hepatic progenitor cell niche, emphasizing similarities and differences in the microenvironments orchestrating regeneration by recruitment of progenitor cell populations or by replication of mature cells.

Abbreviations:
PHx

partial hepatectomy

2-AAF

N-2-acetylaminofluorene

CCl4

carbon tetrachloride

GalN

galactosamine

CDE

choline-deficient ethionine-supplemented

Dipin

1,4-bis[N,N′-di-(ethylene)-phosphamide] piperazine

DDC

3,5-diethoxycarbonyl-1,4-dihydrocollidine

MPK

muscle pyruvate kinase

AFP

α-fetoprotein

dlk

delta-like protein

EGF

epidermal growth factor

HGF

hepatocyte growth factor

TNF

tumour necrosis factor

LPS

lipopolysaccharide

IL-6

interleukin-6

NF-κB

nuclear factor-κB

STAT

signal transducer and activator of transcription

JAK

janus tyrosine kinase

LIF

leukaemia inhibitory factor

OSM

oncostatin M

MAPK

mitogen-activated protein kinase

TGF-α

transforming growth factor-α

FGF

fibroblast growth factor

SCF

stem cell factor

CTGF

connective tissue growth factor

uPA

urokinase-type plasminogen activator

TGF-β

transforming growth factor β

PAI-1

plasminogen activator inhibitor type-1

IFN-γ

interferon-γ

IL-1β

interleukin-1β

LT-α

lymphotoxin-α

LT-β

lymphotoxin-β

SDF-1

stromal derived factor 1

ENA-78

epithelial neutrophil activating protein-78

MIP-2

macrophage inflammatory protein-2

MIG

monokine induced by IFN-γ

IP-10

IFN-γ-inducible protein-10

The regenerative capacity of the adult mammalian liver is immense and reflects a complex physiological response to liver injury during which the remnant organ initiates a series of reactions to promote cell replication and/or growth to restore the functional liver mass. Generation of new cells in the injured adult liver depends on a two-tier cell system comprised of mature, pre-existing liver cells and endogenous liver progenitor cells. Unique to the liver is that pre-existing mature cells constitute the primary option of response to injury, while progenitor cells function as a reserve compartment that is activated when the regenerative capacity of mature cells is compromised. In fact, reconstitution of liver mass after surgical resection is accomplished by progeny of the residual liver cells, including parenchymal hepatocytes, biliary cells and stromal cells that maintain their differentiated phenotype during proliferation. In contrast, injury caused by drugs, viruses, and toxins that impair hepatocyte proliferation results in a transit amplification of progenitor cells, which are used for generation of new hepatocytes and biliary cells. Even though not conclusively established, the consensus is now that the progenitor cells in rodents originate from a hepatic progenitor cell niche harboured in the transitional structures between the hepatocyte canaliculi and bile ducts commonly termed the canals of Hering or terminal ductules, and that a similar progenitor cell niche exists in the human adult liver (1–4). Analogous to most stem and progenitor cells in other organs, the liver progenitor cell has attracted significant attention due to its potential use in future cell therapies for a number of liver diseases, where only whole organ transplantation with subsequent intense immunosuppressive therapy or symptom therapy is the available treatment.

The recent major advances in understanding the mechanisms of liver regeneration derive to a great extent from experimental studies in well-established rodent models, including the use of transgenic and knockout mice. Not surprisingly, accumulating data point to a fundamental role of what could be termed the established hepatic microenvironment. The latter is an intricate cocktail of resident liver cells as well as infiltrating inflammatory cells that express and secrete a variety of growth-modulating factors and their receptors, including molecules as diverse as growth factors, cytokines, and chemokines. Clearly, the cellular and molecular responses to different regenerative stimuli must depend on the injury inflicted and consequently on the molecular microenvironment created in the liver by a certain insult. Therefore, elucidating the responses and subsequently the mechanism(s) that control activation and expansion of a particular liver cell population is a fundamental prerequisite for understanding liver regeneration and the potential development of novel pharmacological and/or transplantation-based approaches for treating life-threatening liver diseases. Recently, excellent reviews on the molecular mechanisms involved in hepatocyte-mediated liver regeneration have been presented (1–4). Here, we will review the current knowledge on molecular responses controlling activation and expansion of the hepatic progenitor cell niche, emphasizing similarities and differences in the microenvironments orchestrating regeneration aided by progenitor cells or mature cells.

MATURE HEPATOCYTES AND BILIARY CELLS AS REGENERATIVE UNITS IN ADULT LIVER

In the normal adult rodent liver, hepatocytes comprise approximately 80% of the hepatic cells. Functions, such as bile secretion, detoxification, metabolism of nutrients, synthesis of vitamins, and production of serum proteins, are all carried out by hepatocytes. These essential functions probably explain, at least in part, the liver's capacity for regeneration. In the resting adult liver, hepatocytes and biliary cells are usually quiescent and rarely undergo cell division. However, if the liver is injured these cells can exit their resting state and replicate to restore loss of tissue mass and function, which in essence represents a process of pure compensatory hyperplasia by residual differentiated liver cells (Fig. 1A). Several models can be used to induce the injury required in rodents. Among the most widely used are partial hepatectomy (PHx) where a portion of the liver is surgically removed, and intoxication with carbon tetrachloride or acetaminophen that induces necrosis of hepatocytes located in the centrilobular areas but leaves hepatocytes in the periportal areas unharmed (Table 1 and references herein). That regeneration is a highly controlled process is partly reflected by the synchronous peak in DNA synthesis at 24 and 40 h for parenchymal cells (hepatocytes) in rat and mouse, respectively, and 12 h later for non-parenchymal cells (biliary, Kupffer, and stellate cells). The liver has regained its mass after approximately 1 week in both species (reviewed in 32). The restoration of liver mass described in rodents is analogous to that occurring in the human liver after surgical resection of part of this organ (3, 4) or after liver transplantation into a recipient larger than the donor (27).

Figure 1.

A. Regeneration from mature cells. In the resting adult liver, hepatocytes and biliary cells are quiescent but upon injury they replicate to restore loss of tissue mass and function. B. Regeneration of the biliary epithelium by de- and/or transdifferentiation of hepatocytes. Upon damage to the biliary epithelium, hepatocytes can de- and/or transdifferentiate to biliary cells and restore the function of the biliary tree. C. Regeneration from progenitor cells. Damage to the hepatocytes blocking their replicative capacity results in activation of the progenitor cell niche located in the canal of Hering. After transient amplification, the progenitor cells differentiate into hepatocytes and biliary cells to aid restoration of tissue loss and function.

Table 1. Hepatocyte- and progenitor cell-mediated liver regeneration
Rat
  1. NB: Only representative publications are listed.

Regenerative processes that depend on replication of mature hepatocytes and biliary cells
• Liver regeneration after partial hepatectomy (PHx) (5)
• Regeneration after carbon tetrachloride (CCl4) (6, 7), or acetaminophen (APAP) (centrilobular) injury (7)

Regenerative processes in which progenitor (oval) cells may generate new hepatocytes and biliary cells
• Galactosamine injury (GalN) (8, 9)
• Allyl alcohol (periportal) injury (AA) (10)
• Choline-deficient diet combined with ethionine (CDE) (11)
• 2-N-acetylaminofluorene intoxication combined with 70% hepatectomy (2-AAF/PHx) (12), carbon tetrachloride injury (2-AAF/CCl4) (13), allyl alcohol injury (2-AAF/AA) (13) or choline-deficient diet (CD/2-AAF) (14)
• Long-term exposure to ethanol (15)

Regenerative processes in which small hepatocyte precursor cells (SHPC) generate new hepatocytes
• Galactosamine injury (GalN) (8, 9)
• Retrorsine intoxication combined with 70% hepatectomy (Ret/PHx) (16, 17)
Mouse
Regenerative processes that depend on replication of mature hepatocytes and biliary cells
• Liver regeneration after partial hepatectomy (PHx) (18)
• Regeneration after carbon tetrachloride (CCl4) (18)

Regenerative processes in which progenitor (oval) cells may generate new hepatocytes and biliary cells
• Galactosamine injury (GalN) (18)
• Allyl alcohol (periportal) injury (AA) (19)
• Carbon tetrachloride (CCl4) injury (20)
• Acetaminophen (APAP) injury (21)
• Choline-deficient diet supplemented with ethionine (CDE) (22)
• 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-supplemented diet (23, 24)
• Long-term exposure to ethanol (25)
• 1,4-Bis[N,N′-di-(ethylene)-phosphamide] piperazine injury combined with 70% hepatectomy (Dipin/PHx) (26)
Human
Regenerative processes that depend on replication of mature hepatocytes and biliary cells
• Live regeneration after partial hepatectomy (PHx) (3, 4) or liver transplantation into a recipient larger than the donor (27)

Regenerative processes in which progenitor cells may generate new hepatocytes and biliary cells (28–31)
• Ductular reactions in advanced stages of chronic hepatitis/cirrhosis of various etiologies
• Fatty liver disease
• Small cell dysplasia
• Massive hepatocyte necrosis

Major loss of the hepatic mass induces about 90–95% of the residing hepatocytes to enter the cell cycle and approximately two doublings of each hepatocyte are required to restore liver mass. However, in some models of liver regeneration differentiated hepatocytes have been observed to replicate at least 70 times (33), leading some investigators to term the hepatocyte a “unipotent progenitor cell” (34). Nevertheless, it has recently been shown that adult hepatocytes can also function as progenitor cells and rescue the biliary epithelium during repair from injury when its proliferative capacity is being compromised (35), indicating that the hepatocyte possesses more plasticity than hitherto recognized (Fig. 1B). This is consistent with the notion that ductular structures can form in the human liver not only by proliferation of pre-existing cholangiocytes or of progenitor cells but in certain cases also through biliary metaplasia of hepatocytes (28, 30), as for instance observed around the centrilobular veins in patients with severe chronic liver stasis due to congestive heart insufficiency or chronic Budd-Chiari syndrome (31, 36).

Confirming the pronounced plasticity of hepatic parenchymal cells is the observation that if cell proliferation after PHx is arrested or if both hepatocyte proliferation and progenitor cell activation are prevented, restoration of liver mass can be achieved by hypertrophy of periportal hepatocytes in the absence of DNA synthesis. This hypertrophic process, which is reversible if the conditions for cell proliferation are re-established, seems therefore to represent an alternative mechanism by which hepatocytes can restore liver mass (37).

PROGENITOR CELLS AS REGENERATIVE UNITS IN ADULT LIVER

Regeneration after injury

The first observation of what today is termed progenitor cells was made in 1937 when small, ovoid cells were reported to appear in livers of rats exposed to the carcinogenic azo dye ‘Butter Yellow’ (38). Twenty years later these cells were termed “oval cells” because of their characteristic morphology with an ovoid nucleus, small size (relative to hepatocytes) and high nuclear to cytoplasmic ratio (39). As originally described, progenitor (oval) cells proliferate in the periportal region of the liver and, as liver damage progresses, they expand transiently and infiltrate into the parenchyma along bile canaliculi between the hepatic cords (39). Subsequently it was reported that these cells encompassed the capacity to restore the lobular architecture in mice fed a methionine-rich diet supplemented with bentonite (40). Although initially considered of little importance, the hepatic progenitor cell niche and the molecular responses that govern its activation have in recent years attracted increased interest. However, the progenitor cell niche in adult liver has proven particularly difficult to study. This can be exemplified by the phenotypic heterogeneity of the transit-amplifying progenitor cell population and many years of controversy regarding its origin. In the expanding literature on progenitor cell-mediated liver regeneration in rodent liver, a number of experimental models have been used (Table 1 and references herein). In the rat, N-2-acetylaminofluorene (2-AAF) treatment in combination with 70% hepatectomy (2-AAF/PHx), 2-AAF treatment combined with exposure to carbon tetrachloride (2-AAF/CCl4), galactosamine (GalN) treatment, and a choline-deficient, ethionine-supplemented (CDE) diet are among the most widely used. In the mouse, 1,4-bis[N,N′-di-(ethylene)-phosphamide] piperazine (Dipin) treatment combined with 70% hepatectomy (Dipin/PHx), the CDE diet, and a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-containing diet have been the most commonly used protocols. Although the actual mechanisms are largely unknown, it is generally believed that the hepatic progenitor cell niche in these models is responding because of impaired hepatocyte proliferation by the injuring agents (Fig. 1C). Yet impaired proliferation does not necessarily provide sufficient stimulation for a true progenitor cell response since a mitogenic stimulus provided by loss of tissue (e.g. PHx or CCl4) is still required after 2-AAF treatment in the rat (13).

Even though not conclusively established, there seems to be a consensus that progenitor cells originate from a hepatic progenitor cell niche in the smaller branches of the biliary tree, including the canals of Hering (Fig. 1C). This is based on convincing studies in the 2-AAF/PHx model of progenitor cell-mediated rat liver regeneration where it was shown that a) the transit-amplifiying progenitor (oval) cells always form ductules elongating as tortuous extensions of the pre-existing canals of Hering terminating at hepatocytes located at the limiting plate (41), and b) the progenitor cell response is not induced when prior bile duct injury has been inflicted by methylene dianiline (42). At the same time, several studies in rats, mice and also humans have indicated that epithelial liver cells, such as hepatocytes, cholangiocytes and progenitor cells, can be generated from extrahepatic bone-marrow progenitor cells in the injured liver (43–48). However, further evidence has shown that the proportion of new hepatocytes generated through this route is very small and the result of cell fusion and not differentiation of haematopoietic stem cells (49–52). Similar recent data have also demonstrated that the hepatic progenitor (oval) cells populating the liver after injury in rodents are derived from endogenous liver progenitor cells and do not arise through transdifferentiation from bone marrow cells (24, 53). Thus, it is currently believed that the contribution of haematopoietic stem cells to the restoration of hepatic epithelial cells after liver damage is not significant (50, 52).

Markers of progenitor cells

A number of protein markers have been used to characterize the transit-amplifying ductular progenitor cell population in adult rodent liver. However, these protein markers are often shared with other cell populations in the adult liver, a phenotypic heterogeneity that is thought to reflect a lineage specification within the population of transit-amplifying progenitor cells. For example, ductular progenitor cells and biliary cells in normal adult rodent liver react equally well with antibodies against a number of proteins, including cytokeratins 7, 8, 18, 19, OV-6 (an anti-cytokeratin 19 antibody), OC.2 (anti-myeloperoxidase) and some other members of the OC series, γ-glutamyl transpeptidase (γ-GT), muscle pyruvate kinase (MPK) and, specific to the mouse, antigen and A6 (Table 2 and references herein), suggesting a specification within the bile duct cell lineage. At the same time, subpopulations of progenitor cells express proteins that are normally only expressed by cells committed to the hepatocytic lineage, including albumin, α1-antitrypsin, hepatocyte nuclear factor 4 (HNF4), and epitopes recognized by the antibody HBD.1 (Table 2 and references herein). Finally, in addition to expressing markers of the hepatocyte or bile duct lineage, transit-amplifying progenitor cells may also share markers with haematopoietic stem cells, including Thy-1, CD34, and c-kit (Table 2 and references herein).

Table 2. Various markers expressed by adult liver progenitor cells
Progenitor cell markersReferences
  1. The first vertical column indicates expression shared with other hepatic cells.
    NB: Only representative publications are listed.

Adult biliary markers
 Glutathione-S-transferase P (GST-P)54
 γ-glutamyl transpeptidase (γ-GT)55
 Cytokeratin 19 (CK19)56,57
 Muscle pyruvate kinase (MPK)58
 Cytokeratin 14 (CK14)56
 OV-6 (recognizes CK14 and CK19)56, 59
 A626, 60
 OC.2 antigen61
 OC.3 antigen61
 Connexin 4362
 c-kit63, 64
 CD3465
 Deleted in malignant brain tumour 1
 (dmbt1)
66
 Stromal-derived factor 1 (SDF1)67
Fetal hepatocyte markers
 α-fetoprotein (AFP)15, 68
 Muscle pyruvate kinase (MPK)58
 Delta-like protein (dlk)69, 70
 Glutathione-S-transferase P (GST-P)54
 γ-glutamyl transpeptidase (γ-GT)55
 Aldolase A and C71, 72
 c-Met73, 74
Adult haematopoietic markers
 Thy-175
 c-Kit63
 CXCR476
 CD3465
Adult hepatocyte markers
 Albumin77
 α1-antitrypsin78
 Hepatocyte nuclear factor 4 (HNF4)79
 HBD.180, 81

To date, only two proteins have been shown to be unique to the transit-amplifying progenitor cell population in the adult rat liver (Fig. 2). The first identified, namely the albumin-related, secreted α-fetoprotein (AFP), is almost certainly the most accepted marker for progenitor cells in the rat and has been reported expressed in several models of progenitor cell-mediated liver regeneration, including the 2-AAF/PHx and CDE protocols (15, 68). While AFP transcripts are highly expressed in the fetal hepatocyte, they are only occasionally detected and at very low levels in cells of the biliary epithelium in normal and hepatectomized adult rat liver (82, 83). However, transit-amplifying progenitor cells expressing AFP are observed after activation of the hepatic stem cell niche in the 2-AAF/PHx and CDE models (15, 68, 84) and the expression of AFP, at least at the transcriptional level, is evident in cells of the terminal bile ductules when exposed to 2-AAF alone, indicating that cells in the hepatic progenitor cell niche do not express AFP unless stimulated (85–87). The second protein identified, delta-like protein (dlk) (69, 70), is a transmembrane protein with several progenitor cell-related functions in other cell systems, among these maintenance of the pre-adipocyte state in adipogenesis, cell-to cell interaction in hematopoiesis, and zonal differentiation in the adrenal cortex (88–91). In progenitor cell-mediated liver regeneration, the expression of dlk is found in cells of the tortuous ductular structures infiltrating deeply into the parenchyma and the connecting small hepatocyte-like cells but is typically absent in ductular structures in the periportal areas (69, 70, 92). The expression of dlk seems to be more restricted to cells committed toward the hepatocytic lineages, which is in agreement with the role of dlk in fetal liver development where it is highly expressed in the hepatocyte but not in the developing bile ducts (69, 70).

Figure 2.

Transit-amplifying progenitor cells in adult rat liver revealed by immunostaining for α-fetoprotein (AFP) and delta-like protein (dlk). A, C, E and G are stained with an antibody against AFP. B, D, F, and H are stained with an antibody against dlk. A and B, normal adult male rat liver; C and D, adult male rat liver 3 days post 70% hepatectomy; E and F, adult male rat liver 9 days post treatment with 2-AAF and 70% hepatectomy (2-AAF/PHx model); G and H, adult male rat liver after 3 weeks on a choline-deficient ethionine-supplemented diet (CDE model). Note the abundance of AFP and dlk positive cells in the 2-AAF/PHx and CDE models where liver regeneration is aided by progenitor cells. Also note the complete absence of this progenitor cell phenotype after 70% hepatectomy when regeneration is accomplished by replication of mature cells (PHx model).

Even though several markers have been reported for the transit-amplifying progenitor cells and their progeny, our knowledge of lineage specification within the hepatic stem cell niche and its progeny is still limited. One explanation is that the majority of progenitor cell markers are intracellular and not surface markers, making the adult progenitor cells difficult to isolate. Although progress has been made using the surface markers dlk (70, 92), Thy-1 and Sca-1 (93) to isolate putative progenitor cell populations from regenerating rat and mouse livers, one of the major problems still to be resolved is the identification of markers or combinations of markers that are more selective or even restricted to the resting putative stem/progenitor cell. However, a first step towards a phenotypic characterization of cells in the putative stem/progenitor cell niche has come from a recent study on the expression of cytokeratin subtypes in the biliary system of the adult rat liver (57). It has long been recognized that a phenotypic feature of mature hepatocytes is the expression of CK8 and 18, whereas CK7 and 19 occur in the biliary epithelium in addition to CK8/18. Surprisingly, the normal adult rat liver, but apparently not the human adult liver, contains a subpopulation of cells forming the smaller branches of the biliary epithelium, including the canals of Hering that only express CK19 in addition to CK8/18. This CK19+/CK7− subpopulation is not present in the liver at birth, but is generated de novo after birth and is preferentially activated by administration of 2-AAF, all features that make this subpopulation of cells prime candidates for the adult hepatic stem cell in rat liver (57).

Progenitor cells in human liver diseases

Hepatic progenitor cells have been observed in several human liver diseases and are therefore not only a rodent phenomenon. Acute zonal or massive hepatic necrosis or chronic liver injury, such as chronic viral, autoimmune or drug-induced hepatitis, primary sclerosing cholangitis, primary biliary cirrhosis, alcoholic and non-alcoholic steatohepatitis, and pediatric liver diseases, such as α1-antitrypsin deficiency, Wilson's disease and extrahepatic biliary atresia, are all examples of pathophysiological conditions that are known to accumulate progenitor cells (30). As seen in rodents, this is caused by injury to and replicative defects of the hepatocytes and/or biliary cell mass. The terms progenitor cell compartment and reactive ductules are used in humans, and are analogous to the often used term oval cell compartment and oval cell ductules in rodents (31). Moreover, “ductular reaction” is generally used to indicate a reaction of ductular phenotype due to the proliferation of cells in pre-existing ductules, activated progenitor cells, and small intermediate hepatocytes (31). Also in human liver, the canals of Hering are believed to harbour the hepatic progenitor niche. The most primitive progenitor cells have the capacity to differentiate down the hepatocytic and biliary lineages through the transitional phenotypical steps of small intermediate hepatocytes and reactive ductules, respectively, which indicates their bi-potentiality. Consistent with that, the human progenitor cells, as those in rodents, express markers characteristic for their fate, with the more primitive cells expressing a mixture of biliary and hepatocytic antigens (often haematopoietic markers as well), the profile of which is subsequently modified during differentiation toward one of the two lineages (30, 31, 94).

Another feature characteristic of the progenitor cell compartment in humans is that the nature of the inflicted injury seems to determine whether intermediate hepatocytes or reactive ductules predominate, as a prevalence of the former cells is observed in diseases with significant hepatocyte damage and loss, whereas proliferation and cholangiocytic differentiation of reactive ductules are more typical of chronic cholestatic biliary diseases (30). Especially in biliary diseases, groups of immature biliary cells have also been detected in the interlobular bile ducts, suggesting that progenitor cells may also participate in the repair of these structures (95). Furthermore, the accumulation and activation of the progenitor compartment has been reported to correlate with the severity of liver diseases (25, 58). For instance, the amount and distribution of intermediate hepatocytes and reactive ductules detected in chronic hepatitis correlates with the degree of intralobular and interface inflammation, respectively (58, 96), supporting a role for cytokines and chemokines secreted by the inflammatory cells in the activation of the progenitor cell compartment (see below). By the same token, the extent of progenitor cell activation in alcoholic and non-alcoholic steatohepatitis is proportional to the stage of the disease, i.e. the degree of fibrosis (25). Furthermore, the extent of progenitor cell activation (ductular reaction) in cirrhosis has been correlated with the progressive exhaustion of hepatocyte proliferative capacity in late stages of this disease (97). Some evidence shows that the reactive bile ductules proliferating in cirrhotic livers are associated with intraseptal hepatocytes, suggesting that the latter are likely to represent newly fomed hepatocytes “budding” from activated progenitor cells (97). Despite many similarities with progenitor cell in rodents, human reactive ductules rarely express AFP, suggesting the existence of some differences between the hepatic progenitor cell compartment in rodents and humans (31).

Finally, the fact that many of the human diseases associated with hepatic progenitor cell activation may result in malignant growth in the liver (98) and the identification of hepatic progenitor cells and progenitor cell antigens in human premalignant and malignant hepatic lesions (99–103) has led to the notion that hepatic progenitor cells may be implicated in the pathogenesis of liver cancer (30, 104). Although it cannot be completely ruled out that progenitor cells activated during human hepatocarcinogenesis solely reflect a regenerative response, further support for their possible carcinogenic role comes from the observation of progenitor cell activation in animal models of chemically induced (105, 106) or transgene-/gene knockout-mediated hepatocarcinogenesis (107–109). In keeping with the pathogenetic role of progenitor cells in hepatocarcinogenesis, many of the experimental protocols employed to activate the progenitor cell compartment are based on carcinogens and indeed are eventually tumorigenic in the rodent liver. While the oncogenic potential of hepatic progenitor cells may represent an important drawback for their potential therapeutic employment in chronic liver diseases, elucidating the molecular mechanisms that underlie their activation and expansion may help prevent this problem and permit safely utilizing their beneficial capacity to regenerate the lost liver mass.

MODULATION OF THE MICROENVIRONMENT IN LIVER REGENERATION: ROLE OF PRIMING, GROWTH, AND GROWTH INHIBITORY FACTORS

Liver regeneration from mature cells is now known to be a delicately regulated and balanced process that involves several cell types, such as hepatocytes, biliary cells, Kupffer, and stellate cells (1, 4). These cells create a microenvironment favourable for regeneration by expressing and secreting multiple growth modulators. It has been suggested that the latter may be divided into factors acting in the priming phase, growth phase, and growth inhibitory phase of the regenerative response, hence the names priming, growth, and growth inhibitory factors (3). It is increasingly evident that some factors and processes in the microenvironment play identical roles in regeneration from mature liver cells and from liver progenitor cells, while others differ significantly. For instance, the absence of progenitor cells during regeneration from mature liver cells suggests that the initial activation processes may differ. This is further illustrated by the fact that hepatocytes and biliary cells “only” need to divide a few times to replace damaged or removed liver mass, whereas progenitor cells need to undergo significant rounds of proliferation and subsequent complex differentiation processes. It is evident that progenitor cells react differently than hepatocytes to injuring factors. Indeed, 2-AAF somehow primes the stem/progenitor cell niche for transit-amplification by a mitogenic stimulus supplied either by loss of liver mass (70% hepatectomy or CCl4 injury) or by infusion of recombinant growth factors, such as epidermal growth factor (EGF) and hepatocyte growth factor, (HGF) while hepatocytes are unable to respond (13, 41, 85, 87, 110).

PRIMING FACTORS

Tumor necrosis factor and interleukin-6

The signalling pathways underlying the early priming phase of liver regeneration are thought to be triggered by the synergistic effect of a wide array of stimuli, including reactive oxygen species, lipopolysaccharides, and inflammatory mediators released into the portal circulation. Lipopolysaccharide (LPS) secreted from the gut and the complement components C3a and C5a, two potent inflammatory mediators of the innate immune response, are essential in liver regeneration mediated by mature liver cells. Both LPS and the complement components act through the induced secretion of priming factors, such as tumour necrosis factor (TNF) and interleukin (IL)-6 from Kupffer cells (111–113). TNF signalling is mediated through interaction of the factor with its two receptors, tumour necrosis factor receptor (TNFR) 1 and 2, expressed on the surface of Kupffer cells and receptor binding activates the downstream transcription factor nuclear factor (NF)-κB. Mice deficient in C3, C5, TNFR1, but not TNFR2, have impaired and abnormal liver regeneration when subjected to 70% hepatectomy (86, 113). Studies have shown that the impaired regeneration process in TNFR1-deficient mice can be rescued by IL-6 treatment, indicating that IL-6 expression is mediated by the TNF signalling in an autocrine/paracrine manner (86, 114). The secreted IL-6 recognises and binds to the IL-6 receptor (IL-6R) binding α chain gp80 on the hepatocytes. Gp80 forms a complex with the gp130 receptor and results in activation of cytoplasmic Janus tyrosine kinases (JAKs), which turn on downstream pathways such as activation and translocation of signal transducer and activator of transcription (STAT) to the nucleus. Subsequently, the translocated STATs function as transcription factors on genes encoding a large number of proliferation-promoting factors, including IL-6 itself, other cytokines, growth factors, and a range of immediate early gene products (115–117).

TNF seems to be equally important in the proliferative response of progenitor cells, both in vitro and in vivo. Observations in rats subjected to the 2-AAF/PHx protocol and treated with the anti-inflammatory agent and inhibitor of TNF, dexamethasone (118), as well as in TNFR1-deficient mice subjected to the CDE protocol have shown that inhibition of TNF signalling results in a reduced progenitor cell response and impaired liver regeneration (86). The Kupffer cells are probably the primary source of TNF during liver injury as pretreatment with a Kupffer cell toxin, gadolinium chloride, ablates the progenitor cell response in bile duct-ligated rats (119, 120). However, the progenitor cell response in CDE-treated TNFR1 knockout mice is not completely suppressed, indicating that other factors with potentially similar functions are involved (86).

Dexamethasone treatment of rats subjected to the 2-AAF/PHx protocol, as mentioned above, impairs progenitor cell response and suppresses expression of TNF. The impaired response can be rescued by IL-6 administration, suggesting that IL-6 has a mitogenic impact on the progenitor cell response (118). This mitogenic effect has been substantiated by in vitro studies where IL-6 has been shown to have a direct effect on progenitor cell proliferation, possibly through the activation of STAT3 (117). IL-6 has been found upregulated during the progenitor cell response in CDE-treated mice and IL-6-deficient mice have impaired progenitor cell response (86). However, even though impaired, the progenitor cell response in IL-6-deficient mice is less affected than in the TNFR1-deficient mice, suggesting involvement of other TNF-inducible mitogenic factors (86). In keeping with this hypothesis, the acute proliferative response of biliary cells to bile duct ligation appears only minimally compromised in IL-6-deficient mice and this has been ascribed to the compensatory role of redundant, IL-6-related cytokines such as leukaemia inhibitory factor (LIF) (121). However, after prolonged bile duct ligation, IL-6 knockout mice showed severe, irreversible cholestasis and inability to compensate for loss of liver mass, suggesting that IL-6-dependent signalling is required in the long-term response to chronic obstructive cholangiopathy (121). That the downstream effectors NF-κB and STAT3 do play important roles in progenitor cell proliferation and differentiation has recently been demonstrated in progenitor cell populations isolated from rat livers treated according to the 2-AAF/PHx protocol on the basis of the expression of the cell surface and differentiation marker OV1. From these studies it has become apparent that transcriptional activities supported by NF-κB and STAT3 are required for progenitor cell activation, expansion, and differentiation. However, an apparently distinct role for these transcription factors at different stages of hepatic stem cell differentiation is indicated and awaits further elucidation (122).

Leukaemia inhibitory factor and oncostatin M

Leukaemia inhibitory factor (LIF) and oncostatin M (OSM) are glycoproteins belonging to the IL-6 family of secreted cytokines, whose components share structural features and pleiotropic biological functions with IL-6. Indeed, although discovered because of their antiproliferative effects on certain tumor cell types, LIF and OSM are also involved in the regulation of development, growth, differentiation and function of haematopoietic, immune and several non-hematopoietic cell systems. Consequently, they participate in tissue repair and remodelling as well as in many inflammatory processes. The actions of IL-6, LIF, and OSM are mediated by binding to specific receptors, i.e. IL-6R, LIF receptor (LIFR), and OSM receptor (OSMR), respectively, that when activated form dimers with the common gp130 signal-transducing receptor, leading to activation of JAKs and STATs as well as MAPK (mitogen-activated protein kinase) cascades (123–125). In addition, OSM is able to signal through LIFR/gp130, reflecting a certain capacity of these cytokines to share and interchange the use of their receptors (124, 125).

Several properties of LIF and OSM suggest that these two cytokines, similarly to IL-6, may be implicated in progenitor cell-mediated liver regeneration. Indeed, LIF and OSM, like IL-6, are capable of stimulating the growth of haematopoietic progenitor cells and inducing the synthesis of hepatic acute-phase proteins, and are well recognized for their capacity to maintain the pluripotential phenotype of mouse embryonic stem (ES) cells in vitro (123). OSM signalling is also known to induce differentiation of hepatocytes during postnatal liver development (126) and appears necessary for hepatocyte proliferation and tissue remodelling during mouse liver regeneration following PHx or carbon tetrachloride-induced necrosis (127). Supporting the view that LIF is involved in the regulation of progenitor cell-mediated liver regeneration, a robust and durable transcriptional upregulation of LIF and LIFR in transit-amplifying progenitor cells is observed in the rat liver treated with 2-AAF/PHx, in contrast to a very transient induction of LIF and LIFR observed in hepatocytes after simple PHx (128). In addition, as mentioned above, LIF appears able to compensate for the lack of IL-6 in the acute ductular proliferative response after bile duct ligation in IL-6-deficient mice (121).

Interesting and conflicting results concerning the role of OSM during the hepatic progenitor cell response have recently been published. Several studies have come to the conclusion that OSM induces growth arrest of both murine and human fetal hepatoblasts and initiates differentiation down the hepatocytic lineage (126, 129, 130). A similar function has been suggested by the analysis of OSM expression during the progenitor cell-mediated regeneration in rats subjected to the 2-AAF/PHx protocol (131). In this case, the OSMR and OSM expression follows the transit amplification of progenitor cells and is characterized by an exclusive localization of OSMR expression to progenitor cells, whereas OSM expression localizes to progenitor cells as well as Kupffer cells. This may implicate the existence of autocrine and paracrine mechanisms of OSM signalling during rat progenitor cell-mediated liver regeneration. In vitro studies have shown that a rat progenitor cell line (OC15-5) as well as primary progenitor cells incubated in a conditioned media from an OSM-transfected cell line stop proliferating and display gene expression patterns as well as morphological changes characteristic for hepatocytes (131). Similarly, putative hepatic progenitor cells can be induced to differentiate in culture by employing growth factors, cytokines, and the support of extracellular matrix components. While HGF promotes the transition of albumin-negative progenitor cells to hepatoblast-like albumin-positive cells, OSM induces the differentiation of the latter to hepatocyte-like cells (132). In contrast, although the expression of OSM and OSMR follows the transit-amplification of progenitor cells in mice fed the CDE diet, OSM does not modulate the growth or maturation state of cultured primary progenitor cells isolated from CDE-fed mice (133). A recent investigation of the expression of LIF, LIFR, OSM, and OSMR proteins in late stages of human cirrhosis has shown that LIF and LIFR are upregulated in biliary cells and reactive proliferating ductules and OSM is upregulated in Kupffer cells, while OSMR, normally expressed at low level in normal human hepatocytes, has not been found expressed at all in cirrhotic livers (134).

Collectively, the findings in rodent models and humans suggest that OSM might be particularly, if not exclusively, required in the adult liver during repair/regeneration to modulate tissue growth and remodelling, since OSM expression has been reported to be low or absent in the normal adult liver of humans and rodents, respectively (129, 134, 135). The results also seem to suggest the existence of species-related differences in the way OSM signalling might be involved in the regulation of progenitor-cell mediated liver regeneration. While in rodents both OSMR- and LIFR-mediated signalling appear implicated (128, 131, 133), the lack of OSMR expression as opposed to the upregulation of LIFR found in human cirrhotic livers has led to the hypothesis that OSM may regulate ductular reactions in human cirrhosis by signalling, in concert with LIF, only through its alternative receptor LIFR/gp130 (134). However, the small number of human liver samples analysed – and the hitherto not excluded possibility that the lack of OSMR expression in cirrhotic livers might reflect technical problems in the methods used to detect this protein – indicates that further work is needed to verify these species-related differences and their significance and to better understand the role played by OSM and LIF in the regulation of the hepatic progenitor cell compartment.

GROWTH FACTORS

HGF, EGF, TGF-α, FGF, SCF, and CTGF

Growth factors such as hepatocyte growth factor (HGF) and transforming growth factor-α (TGF-α) have limited stimulatory growth effect in vivo unless their target cells have been primed, e.g. by IL-6 (3). HGF is produced by mesenchymal cells of various tissues, including non-parenchymal liver cells, and induces pleiotropic effects ranging from stimulation of proliferation, motility (from which the alternative name “scatter factor”), morphogenesis, and in certain conditions survival of normal hepatocytes and extrahepatic epithelial cells to regulation of hematopoiesis, angiogenesis, fibrinolysis and coagulation (136, 137). HGF is secreted as a HGF precursor which is stored bound to the extracellular matrix and when needed cleaved to the active growth factor by proteases, including urokinase plasminogen activator (uPA) and Factor Xa (138, 139). The multiple functions of HGF are mediated by its binding to the membrane-spanning tyrosine kinase receptor c-Met, which is expressed in epithelial cells, including hepatocytes, biliary cells, and progenitor cells (137). By virtue of these properties, the HGF/c-Met system appears an important paracrine and endocrine modulator of mesenchymal-epithelial interactions required during development and repair/regeneration of a variety of tissues, including the liver (136, 137, 140–142). The plasma levels of HGF increase during liver regeneration in rodents and humans and are augmented in patients with chronic liver diseases (3, 136, 143). Ectopic expression of human HGF under the control of albumin regulatory sequences in mouse liver is able to accelerate the rate of liver repair and regeneration after partial hepatectomy (144). However, when administered intravenously or injected directly into the liver, HGF induces very little DNA synthesis unless the liver has been primed by one-third hepatectomy (145), thus categorizing HGF as a mitogenic growth factor without the capacity to “prime” the hepatocyte for replication.

Similarly, the full action of TGF-α and EGF on hepatocytes as growth factors also requires that the liver has been primed by one-third hepatectomy (145). TGF-α, a structurally and functionally EGF-related peptide, is synthesized by hepatocytes as transmembrane pro-TGF-α that in a juxtacrine manner can bind and activate the transmembrane tyrosine kinase receptor for EGF (EGFR) situated on adjacent cells. Pro-TGF-α undergoes extracellular proteolytic cleavage releasing mature soluble TGF-α and binds the EGFR mediating both autocrine and paracrine effects (146, 147). In quiescent liver, most TGF-α exists in a membrane-anchored form, but during liver regeneration secreted forms, including mature protein and partially cleaved variants, are newly synthesized and/or released from cell membranes and extracellular matrix. Among the cells in the body, hepatocytes contain the highest density of EGFR. Despite similar affinity for the EGFR and shared capability of activating the receptor tyrosine kinase activity, TGF-α exerts more potent mitogenic effects on hepatocytes than EGF (147). The expression of TGF-α is highly induced in the hepatocyte-mediated regeneration after 70% PHx, CCl4 or GalN (148, 149), and ectopic, constitutive expression of a TGF-α transgene causes pronounced hepatocyte proliferation and liver enlargement in young mice and an increased hepatocyte turnover in older transgenic mice as compared to control animals (150). However, the fact that a full mitogenic effect of TGF-α as well as EGF when administered intravenously or injected directly into the normal rodent liver can only be obtained in combination with one-third hepatectomy also categorizes these growth factors as mitogenic but without the capacity to “prime” the hepatocyte for replication (145).

Another growth factor that is transcriptionally upregulated during liver regeneration by mature cells is acidic fibroblast growth factor (aFGF) (151), which belongs to the large family of FGFs with high affinity for heparan sulfate proteoglycans of the extracellular matrix. These growth factors bind to and activate one or more members of the family of transmembrane tyrosine kinase FGF receptors (FGFRs), generating several cell type-specific and cell maturation-dependent effects, including proliferation, growth arrest, differentiation or apoptosis (152). In the normal adult rat liver only a low level of FGFR-2 in hepatocytes has been detected, which significantly increases after PHx, whereas the expression of FGFR-1 is only minimally induced (153).

Expression studies have indicated that TGF-α, HGF, and aFGF also play an important role in progenitor cell-mediated liver regeneration. Indeed, all three growth factors and their corresponding receptors are transcriptionally upregulated during the period of the active proliferation and differentiation of progenitor cells in the rat liver subjected to the 2-AAF/PHx protocol (73, 148, 151, 153, 154), and they appear to drive the early proliferation of the progenitor cell compartment (110, 154). Interestingly, the stellate cells, which proliferate concomitantly and in close contact with progenitor cells (41, 105), appear to be the main source of TGF-α (148), HGF (73), and aFGF (151), while the corresponding cognate receptors are strongly expressed in progenitor cells (73, 148, 153, 155), suggesting that the regulation of progenitor cell proliferation and differentiation by the three growth factors occurs primarily in a paracrine manner. In contrast to the predominant expression of FGFR-2 in proliferating hepatocytes after simple PHx, both FGFR-1 and -2 are highly induced during the transit amplification and differentiation of progenitor cells in the 2-AAF/PHx protocol. In particular, FGFR-1 appears mainly expressed in progenitor cells, while FGFR-2 is upregulated in both progenitor and stellate cells (153), suggesting a unique role for FGFR-1 signalling in progenitor cells. The intercellular interaction between stellate cells and progenitor cells is in part mediated by cell-specific membrane heparan sulfate proteoglycans, which are molecules also mediating the binding of growth factors to the receptors and the cellular connections with the extracellular matrix (reviewed in 30).

Urokinase-type plasminogen activator (uPA) is a component of the plasminogen activator/plasmin system, which plays important roles in liver remodelling and regeneration after partial hepatectomy (138, 156). The plasminogen activator/plasmin system has a direct role in migration of cells by cleaving components of the extracellular matrix, e.g. fibrinogen (an acute-phase protein). Furthermore, it has an indirect role in proliferation of cells by activation of growth factors, such as HGF, TGF-α, and TGF-β (138, 147, 157). In the 2-AAF/PHx protocol, expression of uPA, uPA receptor (uPAR), and plasminogen activator inhibitor type 1 (PAI-1) are localized to progenitor cells in ductular reactions with plasminogen activation taking place in the nearby surroundings of the progenitor cells (158). Infusion of HGF and/or EGF, and/or uPA into rats in which the proliferation of ductal and periductal cells is initiated by low dose of 2-AAF alone, has shown that each of these three proteins can expand the population of progenitor cells in vivo, and that this effect is synergistic when they are combined (110). However, at least in this study, HGF and EGF seem preferentially to influence different cell populations, since exposure to 2-AAF combined with infusion of HGF results in proliferation of similar numbers of biliary and stellate cells, whereas infusion of EGF and any combination hereof results in prevalent expansion of biliary cells. In addition to mitogenic effects, infusion of EGF or HGF leads to decreased numbers of cells undergoing apoptosis in response to 2-AAF (110). Collectively, these results indicate that, although 2-AAF acts as priming mitogenic stimulus for putative progenitor cells, the combination of growth factors and uPA is necessary for survival, motility, and expansion of these cells into the liver parenchyma.

The stem cell factor (SCF)/c-kit growth factor/receptor system has also been ascribed an important function during progenitor cell proliferation and differentiation. The growth factor/receptor system is expressed during the progenitor cell response in the 2-AAF/PHx protocol in rat where SCF and c-kit localize to progenitor cells in ductular structures (63, 64). Furthermore, when rats carrying a mutation in the c-kit receptor are subjected to the 2-AAF/PHx model, the overall progenitor cell response is suppressed, suggesting that SCF plays a role in the initial activation of the progenitor compartment (159).

Finally, connective tissue growth factor (CTGF) may play an important role in regeneration from both mature cells and progenitor cells in the adult rat liver. CTGF, a secretory protein and member of the ctgf/cyr61/nov (CCN) protein family, acts on many cell types regulating proliferation, apoptosis, differentiation, angiogenesis, migration, adhesion and extracellular matrix production (160). CTGF expression is increased during liver regeneration following PHx or GalN injury, but also increases in concert with the progenitor cell response in the 2-AAF/PHx protocol. Progenitor cell populations isolated from liver regenerating in response to the 2-AAF/PHx protocol and sorted out by their expression of Thy-1 contain CTGF mRNA, and inhibition of CTGF expression by the synthetic prostacyclin derivative, iloprost, are associated with a significant decrease in the number of proliferating progenitor cells and reduced expression of the progenitor cell marker AFP (161).

GROWTH INHIBITORY FACTORS

TGF-β and activin

In normal mice and rats the liver/body weight ratio is between 4.5–5%. This ratio is also observed when the liver has regenerated following PHx or CCl4 injury, indicating that regeneration is a tightly regulated and well-balanced process. However, knowledge of the underlying mechanisms for the termination process is limited. The TGF-β family of cytokines, such as TGF-β and activin, is likely to play an important role in the modulation and cessation of liver growth (3, 162). TGF-β has anti-proliferative and apoptotic effects on hepatocytes in vivo and in vitro, due to expression of TGF-β receptors by these cells (3, 163–165). The implication of TGF-β in cessation and adjustment of the regeneration process is indicated by its accumulative expression after PHx and the decrease of liver size after infusion of TGF-β (162, 163). In keeping with that, TGF-β knockout mice display a significant increase in the liver/body weight ratio (166). The main source of TGF-β in normal and regenerating liver is non-parenchymal cells, suggesting a paracrine mechanism for preventing excessive hepatocyte replication after PHx (163). However, recent results in mice with disrupted TGF-β signalling suggest that activin A may be the principal factor in the termination of liver regeneration by mature cells (167).

TGF-β has been proposed to play similar modulatory roles in progenitor cell-mediated liver regeneration. In addition to mesenchymal cells, this cytokine is also synthesized by progenitor cells during the early phases of their differentiation (168). Studies conducted in DDC-fed TGF-β transgenic mice, in which hepatocytes produce active TGF-β, reveal impaired progenitor cell response and severely decreased survival, consistent with the notion that TGF-β inhibits the activation of the hepatic stem cells as well as proliferation of hepatocytes (23).

INFLAMMATORY FACTORS

Cytokines: IFN-γ, IL-1β, LT-α and LT-β

Several studies have suggested the involvement of inflammatory factors in liver regeneration, with important distinctions between the process mediated by mature cells and that operated by progenitor cells. One fundamental difference is regarding the influence of the inflammatory cytokine interferon-γ (IFN-γ) on the two types of hepatic regeneration. IFN-γ is a member of the Interferon family of cytokines and is expressed by activated CD4+ T cells, natural killer (NK) cells, and non-hematopoietic cells, such as hepatocytes, in response to other cytokines (for instance, IL-18, IL-12 and IL-1β) (169–172). IFN-γ interacts and binds as a homodimer to cell surface IFN-γ receptors. This interaction causes transactivation of JAKs leading to phosphorylation and dimerization of STATs, which in turn translocate to the nucleus and regulate the expression of a plethora of genes. Several of these target genes in turn encode transcriptional factors or cofactors, thus resulting in many additional genes indirectly regulated by IFN-γ. Together with an intricate modulation orchestrated by signalling pathways dependent on other cytokines, the existence of direct and indirect IFN-γ targets explains, at least in part, the large variety and complexity of IFN-γ biological effects, such as antiviral activity, regulation of innate and adaptive immune responses, antigen processing and presentation, and leukocyte-endothelium interactions, as well as effects on cell proliferation and apoptosis (comprehensive reviews in 173, 174).

In recent years the intriguing and contrasting role played by IFN-γ in liver regeneration by mature cells and progenitor cells has been uncovered. The expression of IFN-γ transcripts is reportedly suppressed in hepatectomized animals (175, 176), though NK cells, an important source of IFN-γ in the liver affected by pathological processes involving immune responses, are activated after PHx and produce IFN-γ (177). Importantly, the infusion of IFN-γ or the endogenous IFN-γ produced by activated hepatic NK cells inhibits hepatocyte-mediated regeneration by upregulating the expression of several antiproliferative proteins (177–179). In contrast, the abrogation of IFN-γ signalling enhances liver regeneration after PHx (177).

While IFN-γ signalling appears to have a negative impact on liver regeneration by mature cells, the effects of IFN-γ on the progenitor cell compartment appear very different. Investigating possible gene networks in the rat 2-AAF/PHx protocol, members of the IFN-γ network, including IFN-γ, IFN-γ receptor β subunit (IFN-γRβ), the IFN-γ-inducing factor IL-18, interleukin-1β-converting enzyme (ICE), intercellular adhesion molecule-1 (ICAM-1), and uPAR, were found to be upregulated during the progenitor cell response and localized to the progenitor cells (180). The intriguing finding that rats treated with 2-AAF alone also expressed the genes of this network, even after a few days, and that only IL-1β and ICE were expressed during hepatocyte-mediated regeneration, indicated that the IFN-γ network was involved in the priming as well as the maintenance of the progenitor cell response (180). Furthermore, the expression of ICAM-1 by progenitor cells also suggests interactions with other cell types, such as lymphocytes, underlining the importance of immune system cells as part of the microenvironment. Consistent with this concept, several studies have shown that stellate cells as well as infiltrating inflammatory cells are in close association with progenitor cells and moreover that the extent of the inflammatory infiltration correlates with the number of progenitor cells in rodents and the extent of ductular reaction in human chronic hepatitis (58, 96, 181). Similarly, a recent time-course analysis in mice fed the CDE diet has shown that progenitor cell activation and expansion correlates with inflammation and cytokine production (182).

In agreement with the notion that IFN-γ is involved in progenitor cell-mediated regeneration, the combination of IFN-γ and TNF or LPS induces the proliferation of rodent liver progenitor cells in vitro, as opposed to the IFN-γ growth-inhibited hepatocytic cell lines (179). In addition, upregulation of IFN-γ and lymphotoxin (LT)-β RNA has been detected during progenitor cell-mediated regeneration in mice subjected to the CDE protocol, with a significant contribution to the overexpression of these transcripts provided by progenitor cells themselves (176). Confirming the importance of IFN-γ signalling for the progenitor cell-mediated regeneration, the progenitor cell response is impaired in IFN-γ-deficient CDE-treated mice (176). In particular, the number of MPK-positive progenitor cells is affected, while progenitor cells expressing A6, a marker for the biliary lineage, are not significantly reduced, possibly indicating a role for the IFN-γ network in the differentiation of hepatic progenitor cells along the hepatocytic lineage (176). However, more work is clearly needed to elucidate the molecular mechanisms by which this pleiotropic cytokine regulates the progenitor cell compartment.

Interleukin-1β is a member of the IL-1 family, which also includes IL-1α, IL-18 and the naturally occurring IL-1 receptor antagonist (IL-1Ra) (183, 184). IL-1β is a multifunctional inflammatory cytokine produced by activated monocytes and macrophages as well as granulocytes (185, 186). IL-1β possesses numerous biological activities, including regulation of inflammatory processes and immune responses as well as induction of acute-phase proteins, due to its ability to induce the expression of other cytokines, transcription factors such as NF-κB, and enzymatic activites, such as cyclooxygenase, phospholipase A2 and inducible nitric oxide synthase (186, 187). IL-1β 1 also modulates cell adhesion and promotes the diapedesis of inflammatory and immunocompetent cells into the extravascular space, exerts angiogenic effects, and stimulates myeloid differentiation of bone marrow stem cells (186).

In the liver regenerating by mature cells, Kupffer cells appear to be an important source of IL-1β, which like TGF-β is thought to play a role in suppressing hepatocyte proliferation and terminating the DNA synthesis induced after PHx (188). Consistent with this notion, selective Kupffer cell depletion abolishes IL-1β expression and enhances hepatocyte proliferation after PHx (189). Moreover, selective pharmacological inhibition of IL-1β has a positive effect on liver regeneration after PHx (190). The implication of IL-1β in the regulation of progenitor cell-mediated liver regeneration has just begun to be unravelled. Cloning based on suppression subtractive hybridization techniques has shown that IL-1β is among the genes associated with progenitor cell proliferation in rat liver subjected to the 2-AAF/PHx protocol (180). In addition, in this experimental model caspase 1/ICE, the enzyme activating IL-1β, is expressed in the ductular structures formed by progenitor cells (180), implying that these cells can participate in the activation of IL-1β secreted by Kupffer cells. These findings are relevant also in light of the capacity of IL-1β to induce IFN-γ expression. Recently, both IL-1β and IL-6 were reported to induce the expression of LT-β in hepatic progenitor cells through activation of NF-κB and other transcription factors, suggesting that IL-1β and IL-6 regulation of progenitor cell activation and/or expansion after chronic liver damage may in part be mediated by LT-β signalling (191). Although, the effects of IL-1β on the hepatic progenitor cell compartment remain incompletely understood, the involvement of IL-1β in regulating IFN-γ and LT-β expression in these cells represents an intriguing aspect that deserves further investigations.

Lymphotoxin-α and -β are members of the TNF family of cytokines that play important roles in organ development, inflammation and immune response by affecting cell survival, proliferation and differentiation (192). When forming homotrimers (LT-α3), LT-α is a ligand for TNFR1 and 2 (p55/p75) alternatively to TNF, while in association with LT-β it forms cell surface heterotrimeric complexes (LT-α1β2) representing the ligand for the LT-β receptor (LT-βR), a TNFR family member (192, 193). LT-β is primarily found in this heterotrimeric form and the ligand complex appears so far to interact exclusively with LT-βR (193). Once bound by LTs, the TNFR and LT-βR signal downstream to activate different isoforms of the NF-κB family of transcription factors, in large part distinct from those activated by TNF-TNFR. In this manner, LT-α and -β induce genes encoding chemokines, integrins and vascular adhesion molecules, thus contributing to attracting and localizing leukocytes to areas of inflammation (192, 194, 195).

Given the critical role of TNFR1 signalling in hepatocyte- and progenitor cell-mediated regeneration (see above), attention has recently been paid to the role of LTs in these responses. Studies using the TNF/LT-α double knockout mouse have indicated that both regenerative units, e.g. hepatocytes and progenitor cells, are hindered in mediating the replacement of lost or damaged tissue mass when signalling via TNFR1 is abolished (196). However, similarly to IFN-γ, LT-β may play quite different roles in liver regeneration by mature cells or progenitor cells. Indeed, while a simple PHx downregulates LT-β mRNA expression in mouse liver, LT-β and LT-βR are upregulated in the progenitor cells proliferating in CDE-treated mice (176). In addition, LT-β and LT-βR knockout mice display a decreased number of MPK- and A6-positive progenitor cells in response to the CDE diet (176), suggesting an active role of LT-β-mediated signalling in the expansion of these cells. Kupffer cells are likely to be another important source of LT-β during progenitor cell proliferation, as these cells transcriptionally upregulate LT-β after bile duct ligation (197). A possible role for LT-β in modulating progenitor cell-mediated liver regeneration has also been suggested by observations in human patients with chronic hepatitis C, where LT-β expression localizes to reactive ductules, small portal hepatocytes and inflammatory cells, and seems to correlate with the extent of fibrosis (198).

Chemokines: SDF1, IP-10 and MIP-2

Chemokines are a superfamily of structurally related chemotactic cytokines comprising over 50 members that are synthesized and released by inflammatory cells, such as monocytes/macrophages and polymorphonuclear neutrophils. They exert diverse biological functions, including recruitment of leukocytes to tissues during homeostasis or inflammation, coordination of innate and adaptive immunity, and regulation of angiogenesis and organogenesis (199–201). Evidence has been provided for the involvement of chemokines and chemokine receptors in the pathophysiology of several inflammatory and/or autoimmune diseases, including certain hepatic diseases (199, 200). Chemokines are divided into four subgroups based on the location of their invariant cysteine (C) motifs near the N-terminal portion of the molecule. The motif can be positioned as CC, CXC (X being any amino acid) C or CX3C (199). The CXC group is further divided into ELR+/ ELR− (an amino acid motif) chemokines. Chemokines can act synergistically with other chemokines or cytokines to produce a given biological effect, also when the concentrations of the single chemokines per se would be inadequate to induce a given response (202). Their complexity is further substantiated by the affinity of individual chemokines for more than one chemokine receptor. The chemokine receptors all belong to the 7-transmembrane G-protein-coupled receptor family with the amino and carboxylic terminal domains localized extracellularly and intracellularly, respectively. This conformation ensures coupling of extracellular ligand binding to intracellular heterotrimeric G proteins. At present, 16 chemokine receptors have been reported. These are divided into four subgroups and classified by their cysteine motif in the N-terminal segment: CC-receptors (CCR1–9), CXC-receptors (CXCR1–5), C-receptor (XCR-1), and CX3C-receptor (CX3CR1) (200).

Accumulating data suggest important functions for chemokines and chemokine receptors in the process of liver regeneration mediated by mature cells. CXC ELR+ chemokines, such as ENA-78 (epithelial neutrophil activating protein-78), MIP-2 (macrophage inflammatory protein-2), and IL-8, have a mitogenic effect on hepatocytes in vitro and have been found significantly elevated in rat liver after PHx (203). Moreover, neutralization of ENA-78 or MIP-2 slows the rate of liver regeneration (203), suggesting that CXC ELR+ chemokines have positive effects on hepatocyte proliferation after acute liver injury. In keeping with this notion, administration of exogenous CXC ELR+ chemokines enhances hepatocyte proliferation after PHx (204) and facilitates the regeneration and repair of mouse liver after severe paracetamol-induced injury (205). The IFN-γ-inducible CXC ELR− chemokines MIG (monokine induced by IFN-γ) and IP-10 (IFN-γ-inducible protein-10) are significantly elevated in hepatocytes stimulated with IFN-γin vitro (206). In vivo studies have shown that IP-10 is induced in mouse liver in response to PHx, bile duct ligation, CCl4 or GalN intoxication (18). IP-10 is expressed prior to inflammation with a bimodal pattern peaking just before maximum DNA synthesis in parenchymal and non-parenchymal cells after PHx, which is characteristic for immediate early genes. Furthermore, dramatic upregulation of IP-10 is observed in TNF-α, but not IL-6-treated hepatectomized mice (18). Collectively these results indicate that IP-10 could be a chemokine that links tissue injury and inflammation with regeneration (18).

Recent investigations have addressed the role of stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 in the rat liver regenerating via activation of the progenitor cell compartment (67, 76). SDF-1 is a member of the CXC ELR− chemokine family that binds to the CXCR4 receptor, as a unique ligand. The receptor CXCR4 is expressed by most leukocyte populations, endothelial cells, as well as by epithelial cells, while its ligand SDF-1 is expressed in a broad range of tissues and is a potent chemoattractant for a variety of cells, including haematopoietic stem cells, lymphocytes, and monocytes (200, 207). Targeted genetic ablation of the genes encoding SDF-1 or CXCR4 has revealed an essential role of the SDF-1/CXCR4 axis during embryogenesis and organogenesis (208). This axis might be involved in the differentiation of progenitor cells to a more mature form, which is illustrated by the supporting role of SDF-1 during B-lymphopoiesis and by the observation that, in epithelia renewing from precursor cells such as the intestine, CXCR4 is expressed by the less differentiated cells (209, 210). In the rat liver regenerating by activation of progenitor cells, the increase in SDF-1 expression correlates with the accumulation of liver progenitor cells. In contrast, SDF-1 is not expressed during hepatocyte-mediated regeneration (76). Immunohistochemistry localized expression of CXCR4 and SDF-1 to progenitor cells and hepatocytes, respectively. This was interpreted to indicate that progenitor cell activation and migration into the parenchyma was achieved using a hepatocyte-generated SDF-1 gradient, since progenitor cells migrated along a SDF-1 gradient in in vitro chemotaxis assays (76).

However, these results have been challenged by a later study reporting that the expression of SDF-1 indeed correlates with the liver progenitor cell response, but that SDF-1 localizes to the biliary cells lining the bile ducts, single progenitor cells, and progenitor cells forming ductular structures, but only rarely to hepatocytes (67). SDF-1 was also detected in the biliary cells lining interlobular bile ducts in control and hepatectomized rats. CXCR4 expression followed the progenitor cell response and in situ hybridization localized CXCR4 mRNA to the cytoplasm of single progenitor cells as well as periportal progenitor cells forming ductular structures, but not biliary cells lining the bile ducts. Interestingly, double staining for the CXCR4 transcript and AFP revealed that these co-localize in some periportal progenitor cells, forming ductular structures, but more interestingly that the CXCR4 transcript is predominantly localized to progenitor cells in the immediate periportal area, whereas AFP is found more distally. Finally, neutralization of SDF-1 activity decreases the proliferation of progenitor cells, suggesting that the SDF-1/CXCR4 axis is involved in the activation and early expansion of the endogenous liver progenitor cell compartment (67). In support of this hypothesis is a similar study in pancreas regeneration pointing to the SDF-1/CXCR4-dependent signalling as an important player in stimulating survival, proliferation, and migration of pancreatic ductular progenitor cells (211).

CONCLUDING REMARKS

At present, orthotopic liver transplantation is the treatment of choice for many inherited and acquired liver diseases. Although recent developments have meant that the alternative –achieving liver repair through transplantation and/or activation of endogenous hepatic cells with stem-like properties – holds great promise, several aspects require further investigation. First of all, a better understanding of the mechanisms responsible for development of tissue damage, along with the cellular and molecular mechanisms allowing surviving stem/progenitor cells to proliferate and generate new hepatocytes and cholangiocytes in a hostile environment, is needed. Secondly, the identification of new markers specific for different populations of hepatic progenitor cells is warranted, in order to isolate and characterize these cells and subsequently transplant them and test their regenerative capacity in vivo. Finally, elucidating the responses and subsequently the mechanisms that control activation and expansion of a particular liver progenitor cell population is a fundamental prerequisite not only for understanding liver regeneration but also for developing novel pharmacological, genetic and/or transplantation-based therapies for life-threatening liver diseases.

This work was supported by the Danish Medical Research Council (grants 22-03-0277 and 2052-01-0045 to the Danish Stem Cell Research Centre), Savværksejer Jeppe og Hustru Ovita Juhls Mindefond, Gerda og Aage Haensch's Fond, Augustinus Fonden, Aase og Ejnar Danielsens Fond, Beckett-Fonden, Direktør Jacob Madsen & Hustru Olga Madsens Fond, and the LEO Foundation.

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