Recent advances in our understanding of the basic mechanisms that control liver regeneration and repair will produce the next generation of therapies for human liver disease. Insights gained from large-scale genetic analysis are producing a new framework within which to plan interventions. Identification of specific molecules that drive regeneration will increase the options for live-donor liver transplantation, and help treat patients with small-for-size syndrome or large tumors who would otherwise have inadequate residual mass after resection. In a complementary fashion, breakthroughs in the ability to manipulate various cell types to adopt the hepatocyte or cholangiocyte phenotype promise to revolutionize therapy for acute liver failure and metabolic liver disease. Finally, elucidating the complex interactions of liver cells with each other and various matrix components during the response to injury is essential for fabricating a liver replacement device. This focused review will discuss how a variety of important scientific advances are likely to impact the treatment of specific types of liver disease.
Liver transplantation for end-stage liver disease is a great human technological achievement. Despite this success, patients are still dying on the waiting list, and advances in the treatment of liver disease are urgently needed. In few clinical scenarios is the line between success and failure as sharp as between a liver that regenerates and repairs itself and one that does not. Successful regeneration often leads to normal liver function without sequellae, whereas failed regeneration results in death unless a transplant can be performed. It follows that methods to stimulate liver regeneration and repair would have profound implications.
Pharmacologic enhancement of liver regeneration would have a broad impact. For example, if a live-donor operation could remove the left-lateral segment, and the recipient treated with medication to enhance of liver growth, it would obviate the need for the complication-ridden 50 or 60% hepatectomy currently necessary for adult-to-adult live-donor transplants (1). In patients with small-for-size syndrome, stimulation of liver regeneration would be life-saving (2). This ability would also allow more extensive liver resections in patients with hepatoma who would otherwise have inadequate residual mass. Concomitant with benefit to the patient, waiting times would decrease by allowing tumor patients to come off the list.
Development of therapies that harness the ability of various cell types to adopt a liver-specific fate similarly has far-reaching implications. In particular, patients with fulminant liver failure would benefit from repopulation of the liver. Patients with metabolic disorders could be treated by providing cells with normal enzymatic function. Auxiliary liver transplantation and the development of liver-assist devices would be enhanced with cell-based treatments.
Understanding the essential interactions between the parenchymal cells in the liver and nonparenchymal cells and the extracellular matrix (ECM) during regeneration is essential for strategies seeking to develop a fully artificial liver. Achievement of this goal will provide virtually limitless expansion of the patients with liver disease who can be successfully treated.
This targeted review will discuss recent advances in topics related to liver regeneration and repair, and how they are likely to lead to therapeutic interventions.
Large-Scale Genetic Pathways During Liver Repair
Role of developmental processes
Large-scale transcriptional analysis identifies broad themes during liver repair, and forms a guide for designing therapies. These studies, made possible by recent technological advances, are producing fundamental changes in our thinking about how the liver repairs itself. As a first question, it is interesting to consider to what extent liver regeneration is true regeneration at the molecular level. In other words, does the regenerating liver recapitulate the gene expression profile of the developing liver?
At a molecular level, liver regeneration is surprisingly distinct from liver embryogenesis. Multiple observations support this conclusion. At the large scale of chromatin structure, which influences transcriptional activity, data from our laboratory have shown that proteins that modify chromatin structure in the embryonic liver are not induced during regeneration. Furthermore, the vast majority of transcription factors known to be essential for normal liver development are not induced after partial hepatectomy. These include genes necessary for hepatocyte differentiation, migration of primitive hepatocytes, hepatocyte proliferation and the inhibition of hepatocyte apoptosis. In fact, we have shown that embryonic liver development and liver regeneration are most alike in that both tissues have a proliferative signature, a trivial and expected similarity (3).
Although the large-scale transcriptional signature of the regenerating liver does not recapitulate development, some developmental pathways are coopted during regeneration. For example, Forkhead box (Fox) and Notch signaling seem to have a role both during development and during regeneration. Loss of FoxM1 during hepatogenesis impairs intrahepatic bile duct development, and during liver regeneration impairs the proliferative response (4,5). Similarly, Notch signaling plays dual roles. Notch is a transmembrane receptor found nearly ubiquitously in vertebrate tissues. It has numerous ligands including another transmembrane protein, Jagged-1. In embryos, bile duct formation requires Jagged-1; mutations cause Alagille syndrome. In adults, hepatocytes and biliary cells express Notch and Jagged-1, and RNA inhibition of Notch signaling suppresses liver regeneration (6). These results suggest some limited recapitulation of embryonic signaling pathways during regeneration.
These data are consistent with the idea that at the molecular level, liver regeneration is not ‘true regeneration’ but rather hepatocyte hyperplasia. The implication of these findings is that there may be fairly straightforward and simple ways based on signaling pathways to enhance regeneration in patients with inadequate liver mass or to induce liver formation in artificial devices.
Transcriptional programs and paradigms
Next, it is important to identify the transcriptional signature of the regenerating liver to learn about its priorities. This knowledge will be useful in supporting the liver as it regenerates. Restoration of liver mass after injury is a highly coordinated event in which functional classes of molecules are transcribed in a highly synchronized fashion. For example, Arai and colleagues demonstrate genes involved in protein synthesis and posttranslational processing are upregulated early and in a sustained fashion. In contrast, plasma proteins and intermediate metabolism are induced later (7). White and colleagues demonstrate that lipid, fatty acid and hormone metabolism genes are downregulated quickly after hepatectomy, and remain that way through the first round of cell division. In contrast, transcription of genes important for ribosome biogenesis, protein biosynthesis and cytoskeletal assembly occurs by 16 h and remains at high levels through the first round of hepatocyte proliferation (8). These data suggest that the liver is most interested in protein synthesis early after hepatectomy, presumably to allow it to restore its mass and manage its huge metabolic load despite a reduced cell mass. These findings also suggest that metabolism of lipids and fatty acids is a secondary priority for the damaged liver. These are valuable insights that may identify particular functions of the liver that need to be addressed to enhance regeneration in the acute period.
Enhancing Liver Regeneration— Perspectives and Historical Achievements
Even a small increment of increased liver regeneration has the potential to transform mortality into a complete cure in cases in which liver mass is insufficient. Experimental models provide hope that stimulation of liver regeneration can improve outcomes. For example, blockade of the receptor for advanced glycosylation endpoints, which enhances liver regeneration, also improves survival after massive hepatectomy (9). Agents and procedures able to influence regeneration belong to many categories, and recent advances greatly expand the number of potential therapeutic options.
The original description of successful two-third hepatectomy in rodents in the 1930s and reports in the late 1940s of enhanced hepatocyte proliferation in the parabiotic partner of an hepatectomized rat mark the beginning of the modern study of liver regeneration (10,11). Building on these findings, various researchers identified many elements necessary for normal regeneration. These include portal blood flow, insulin and glucagon production by the pancreas, the ratio of a soluble factor to the size of the liver remnant, glucose metabolism and plasma amino acid composition (12–16).
In a particularly brilliant series of technical studies, Starzl and colleagues performed the Whipple procedure in dogs, followed by reconstruction of the superior mesenteric vein into the left hepatic lobe, and the splenic vein and its residual pancreatic flow into right lobe. Preferential growth of the right lobe, which received pancreatic effluent, pointed to a role for pancreas-derived factors in liver regeneration. Follow-up studies in which direct infusion of insulin into the left lobe was mitogenic for hepatocytes implicated insulin as the relevant factor (15,16). Later studies demonstrated the ability of branched chain amino acids to enhance liver regeneration (17), suggesting normal plasma amino acid profiles are critical for regeneration. These investigations began to paint a picture of liver regeneration as a complex process with many inputs and influences.
Modern understanding of liver regeneration and repair focuses on crucial roles for cytokines, transcription factors and growth factors.
Among cytokines, both tumor necrosis factor (TNF) and interleukin-6 (IL-6) are key molecules. Though not directly mitogenic for hepatocytes, plasma levels of TNF increase after hepatectomy and TNF potentiates the effect of growth factors including hepatocyte growth factor (HGF) (18). Decreased TNF signaling, achieved using antibodies or genetic deletions of receptors, delays hepatic regeneration (19,20). IL-6, produced by Kupffer cells, is similarly necessary for normal liver regeneration (21). It may act as a direct hepatocyte mitogen and also help to manage the acute-phase response of hepatocytes (22–24).
Transcription factors including the AP-1 complex and STAT-3 are essential for normal liver regeneration (see Reference 25; reviewed in Reference 26). DNA binding activity of AP-1 increases dramatically in response to partial hepatectomy (27). Liver-specific loss of either c-Jun, part of the AP-1 complex or STAT-3 impairs liver regeneration after partial hepatectomy (28,29).
Hepatocyte growth factor (HGF) and epidermal growth factor (EGF) are critical for the regenerative response. HGF is weakly mitogenic for hepatocytes, likely requiring hepatocyte priming to drive proliferation (30). In rats, circulating EGF, likely from the salivary gland, is required for normal regeneration (31,32).
These factors or their analogues, alone or in combination, are all potential therapeutic targets.
Enhancing Liver Regeneration—New Paradigms: Bile acids
Despite being intimately associated with liver physiology and metabolism, a role for bile acids in liver regeneration has only recently been appreciated (33). In these studies, mice fed bile acids to increase serum levels demonstrated enhanced liver regeneration, whereas mice fed bile binding resins to decrease serum levels demonstrated inhibited regeneration. In a series of elegant experiments, this response was found to be mediated by the primary nuclear bile acid receptor FXR. This finding opens up the possibility of modulating the regenerative response by adjusting bile concentrations.
Transplantation of cells to take up residence in the liver is relevant for a number of specific clinical problems. This technology has the potential to enhance the capacity of the liver to recover from massive injury as might occur in acute liver failure. Also possible is to repair inborn errors of metabolism without having to replace the entire liver mass. Other applications include cell transplantation as primary therapy or as an adjunct to auxiliary liver transplantation (34). Finally, these strategies may be useful in helping to populate an artificial liver replacement device. Recent discoveries concerning the origin of hepatocytes and potential stem cell populations are critical to addressing these issues. They define a number of possible progenitor populations that may be useful as sources for cells in the liver (Figure 1).
In healthy animal livers after partial hepatectomy, most new hepatocytes come from old hepatocytes (35). In disease states or under particular stress, a variety of cell types have the capacity to replenish the hepatocyte pool or contribute to its genetic material, either via transdifferentiation (from cells normally of other lineages) or differentiation (from progenitor or stem cells). The extent to which old hepatocytes themselves replenish the pool in humans is unclear. Since much clinically relevant regeneration in humans occurs over a long period of time in chronic disease, the origin of hepatocytes in humans is more difficult to study compared with acute injury models in animals. It may be that transdifferentiation or differentiation is the dominant mode of hepatocyte replacement in certain disease states.
The regenerative capacity of human liver cells in vivo appears to be, for practical purposes, unlimited. In mice engineered so that their hepatocytes destroy themselves by expressing the toxic urokinase-type plasminogen activator, infusion of human hepatocytes repopulates the entire mouse liver (36). Transplanted hepatocytes can double at least 69 times (37), and so hepatocyte transplantation has great appeal in aiding liver regeneration.
Direct transplantation of adult hepatocytes can be used to increase liver mass or address metabolic deficiencies. The viability of this approach was confirmed in the mid 1970s with the demonstration that isolated hepatocytes could lower the bilirubin in rats with UDP-glucuronosyl transferase deficiency (Crigler–Najjar syndrome type I) (38). Follow-up human studies confirmed that transplantation of mature hepatocytes could improve the manifestations of Crigler–Najjar syndrome (39), ornithine transcarboxylase deficiency (40), peroxisomal biogenesis defect (41) and glycogen storage diseases (42).
An interesting twist on the origins of hepatocytes came from reports in the early 2000s that bone marrow could transdifferentiate into hepatocytes and repopulate the liver. Females undergoing bone marrow transplantation from male donors and male recipients of livers from female donors both demonstrate the presence of the Y chromosome in hepatocytes (43). These results suggested that bone marrow might be a source of hepatocytes. Taken together, these reports suggested a scenario in which bone marrow cells were taking up residence in the liver and transdifferentiating into hepatocytes as an ongoing process.
In a series of carefully performed studies, however, the genetic material in the liver derived from the bone marrow was unequivocally found to be due to fusion of marrow cells with hepatocytes (44,45). It is likely hematopoietic cells generally do not contribute to the hepatocyte pool unless there is a strong selection pressure for this to occur (46). On the other hand, this ability of bone marrow cells to act as a vector for genetic material may have therapeutic implications. A critical study demonstrated that mice with hereditary tyrosinemia due to a deficiency in fumarylacetoacetate hydrolase could be cured with bone marrow from donors containing the enzyme (47).
Another group of endogenous cells that may contribute to liver regeneration are oval cells. These small, rounded cells appear in the portal region of the lobule during chronic injury or in certain models of acute injury in which hepatocyte proliferation is inhibited. They express markers of early hepatocyte lineage (α-fetoprotein), as well as markers of mature hepatocytes (albumin) and biliary cells (CK-19) (48). In contrast to embryonic stem cells that can differentiate into cells of all germ layers, the oval cells lineage seems to be limited to hepatocytes and biliary cells. In an important report on oval cells using a variety of tracking methods, Wang et al. demonstrated that oval cells are an intrahepatic progenitor cell capable of differentiating into both mature hepatocytes and bile duct epithelial cells (49). They are able to repopulate the liver after injury, and are much more easily cultured than hepatocytes. Oval cell proliferation seems to be a characteristic of certain human disease, including primary biliary cirrhosis (50,51). These studies suggest that oval cell transplantation could be a therapeutic tool.
Multipotent stem cells
The role of true, multipotent stem cells during regeneration is unknown, but these cells have therapeutic promise. Recent work has established that embryonic stem cells might provide a mechanism to produce large numbers of hepatocytes for transplantation (52). To demonstrate the utility of this approach, human hepatocytes derived from stem cells have been shown to repopulate the livers of mice with severe combined immunodeficiency (SCID) (53).
Fetal liver cells
An alternative approach to repopulating the liver transplants fetal liver cells harvested from embryonic liver. These cells can generate both cholangiocytes and hepatocytes (54). Human liver progenitor cells can proliferate for several months in culture, with each cell undergoing more than 40 divisions and retaining a normal karyotype. These progenitor cells differentiate into mature hepatocytes in mice with SCID, integrating in the liver parenchyma and proliferating following liver injury (55). Reflecting a diverse potential, transplantation of fetal liver cells may even be used as a source of stem cells (56).
Interplay Between Hepatocytes, Other Cell Types and Platelets
A number of non-parenchymal cells populate the liver, including resident macrophages (Kupffer cells), endothelial cells and stellate cells. They interact with hepatocytes in complex ways to control liver regeneration. Understanding these interactions opens therapeutic opportunities, and is critical for designing liver replacement devices.
Kupffer cells are required for normal liver regeneration. They secrete IL-6 that may be a direct hepatocyte mitogen and increases the production of HGF from other liver cells (21,57). Endothelial cells themselves produce HGF, which is mitogenic for hepatocytes. In response to HGF, hepatocytes produce platelet-derived growth factor that in turn is a mitogen for endothelial and stellate cells (58). In this way, a feedback loop is created to balance the responses of different cell types (Figure 2).
Stellate cells are also critical for liver regeneration. They are of the myofibroblastic lineage and reside adjacent to hepatocyes, producing HGF as well as transforming growth factor-β (TGF-β) (59). Interestingly, since TGF-β inhibits hepatocyte hyperplasia, stellate cells may serve as a check on regeneration, either limiting its pace or helping to arrest growth after the liver has grown to sufficient size. Limiting hepatic growth after adequate regeneration may be equally complex as the regenerative response itself.
The importance of cell interaction in the liver is demonstrated by the distinct effect of various mitogens under different experimental conditions. When added to hepatocyte cultures, vascular endothelial growth factor (VEGF) is not mitogenic. In contrast, when administered to whole animals, VEGF causes proliferation of endothelial cells and hepatocytes. Since the VEGF receptor VEGFR1 is present on endothelial cells, it is likely that in vivo, VEGF activates signaling pathways in endothelial cells that then produce HGF to stimulate hepatocyte proliferation (60). In a similar manner, HGF is a mitogen for hepatocytes only under certain circumstances, suggesting that the effect of HGF itself is dependent on other interactions (Figure 2).
Synchronous with an increase in the number of hepatocytes that occurs during regeneration, an increased endothelial mass must be created to service the new hepatocytes. This fact is critical to designing the next generation of technologies for liver replacement. In a series of important investigations, Beldi and colleagues examined the vascular endothelial response after hepatocyte injury, identifying a critical role for purinergic signaling. This pathway is driven by extracellular nucleotides derived from platelets. Signal transduction occurs after successive hydrolysis of ATP and ADP to AMP and ultimately adenosine, a process dependent on the vascular ectonucleotidases CD39 and CD39L1. These enzymes are induced on the endothelial cell surface after injury (Figure 2)(61). Proper functioning of CD39 is necessary to facilitate HGF release via a VEGF-mediated paracrine mechanism. In mice with targeted disruption of CD39, the response to liver injury is blunted with decreased hepatocyte and endothelial proliferation (62). These studies help to establish a direct link between the endothelial response and the platelets that are intimately associated with them during injury.
Platelets have additional effects on liver regeneration. Lesurtel and colleagues recognized the close interaction of platelets with endothelial cells at the time of injury and demonstrated a direct effect of platelets on liver regeneration. When Busulfan or platelet-specific antibodies were used to deplete platelets, or clopidrogel was given to prevent platelet aggregation, liver regeneration was impaired. These authors went on to identify serotonin as the responsible platelet-derived factor (63).
Other studies establish the therapeutic potential of platelets. Mice with thrombocytosis demonstrate enhanced liver regeneration (64). In coculture experiments using both platelets and hepatocytes, hepatocyte proliferation is enhanced by platelet-derived HGF and IGF-1 (65), suggesting that platelets may directly secrete hepatocyte mitogens.
Role of Matrix
Construction of a bioartificial liver for transplant will almost certainly have to address the issue of fabricating the proper scaffolding or stroma. Rather than serving as a passive foundation, ECM and factors within it play an active role in controlling a variety of cellular responses of the regenerating liver. It is likely that growth factors are bound by and sequestered in the ECM, awaiting activation during an injury (Figure 3). In a series of papers, Michalopolous and colleagues demonstrated that restoration of liver mass after injury is associated with profound alterations in ECM. These studies predict that release of mitogens bound to the ECM into the extracellular space is necessary for liver regeneration (66,67). Mice with targeted deletion of MMP-9, a metaloproteinase important for matrix remodeling validate this hypothesis. These mice fail to elevate HGF and VEGF, and display delayed hepatocyte proliferation after partial hepatectomy (68). The mechanism of this can be deduced from the following observations: (1) HGF is known to bind hepatic ECM and be released in the event of matrix remodeling (69), and (2) in wild-type mice, plasma HGF increases 1 h after hepatectomy, which precedes the rise in mRNA that occurs a few hours later (70). Taken together, these findings suggest a model in which HGF is stored in the ECM. In response to injury, matrix metalloproteinases including MMP-9 produced in Kupffer cells are released into the ECM. This releases growth factors sequestered in the matrix (71) (Figure 3).
A similar mechanism may exist for VEGF. Multiple isoforms of VEGF bind the heparin and heparan sulfate proteoglycans on the ECM (72). Interestingly, binding to ECM stabilizes and protects VEGF from proteolysis. Matrix remodeling then liberates VEGF and activates its signaling (73). In this way, the liver maintains a latent reservoir of growth factor that requires only matrix injury or degradation to release.
There may be another mechanism via which matrix regulates regeneration. In a model of toxic liver injury, Bezarra and colleagues demonstrated that tPA and uPA are critical to physically clearing out debris necessary to allow hepatocyte proliferation (74). These findings speak to the physical requirements of the liver stroma to act a scaffold to support hepatocytes proliferation (Figure 2).
Taken together, these findings provide guidance for the development of the scaffold or architecture underpinning bioartificial liver devices (75).
Despite the tremendous achievements in our ability to treat end-stage liver disease, the need for further therapies remains pressing. Harnessing the potential of the liver to repair and restore itself after injury promises to impact a wide variety of liver disease as an adjunct or alternative to transplantation. Diseases particularly amenable to being affected by these advances include problems of inadequate residual liver mass, fulminant liver failure and the fabrication of artificial liver devices. These hurdles are likely to be overcome as our understanding increases.
The author would like to thank Dr. Douglas Hanto and Dr. Simon Robson for critical review of the article.