Models of Liver Regeneration

In the past 50 years, much has been learned about what initiates liver regeneration, how it is sustained, and what inhibits it. Liver regeneration is most clearly shown in the experimental model that was pioneered in 1931 by Higgins and Anderson. In this model, a simple two-thirds partial hepatectomy (PHx) was performed, without damage to the lobes left behind. This led to enlargement of the residual lobes, which made up for the mass of the removed lobes in 5 to 7 days. Other well-known models of liver regeneration are associated with extensive tissue injury and inflammation and include the use of hepatic toxins—such as ethanol (EtOH) (Kaplowitz, 2002), carbon tetrachloride (CCl₄) (Reynolds, 1963), and galactosamine (GalN) (Dabeva & Shafritz, 1993)—bile duct ligation (Kountouras et al, 1984) or portal vein ligation (Bilodeau et al, 1999), and I/R injury (Jaeschke, 1998). Newer models include transgenic albumin promoter urokinase-type plasminogen activator (u-PA) fusion constructs (Heckel et al, 1990) and PHx in Zebrafish (Kan et al, 2009). In each model, different toxic agents injure specific liver cell subpopulations; PHx is therefore the preferred in vivo model to study the regenerative response. It has also been demonstrated that regenerative signaling observed in a rat liver transplant model of I/R injury is similar to that observed after PHx (Debonera et al, 2001).

General Features of Liver Regeneration

Liver regeneration after surgical resection is carried out by proliferation of existing mature cellular populations. These include hepatocytes, biliary and fenestrated endothelial cells, Kupffer cells, and cells of Ito (stellate cells; Fig. 5.2) (Gressner, 1995). The kinetics of cell proliferation and the growth factors produced by proliferating hepatocytes suggest that hepatocytes provide the mitogenic stimuli that lead to proliferation of the other cells; the degree of hepatocyte proliferation is directly proportional to the degree of injury (Bucher & Swaffield, 1964).

FIGURE 5.2 Pathways of liver regeneration initiated by major hepatectomy. After hepatectomy, nonparenchymal cells—stellate cells, Kupffer cells, leukocytes, and platelets—are activated by soluble factors. As a result, Kupffer cells release tumor necrosis factor-α and interleukin-6. The cytokines cause a priming of the remnant hepatocytes, and concurrently, extracellular proteases, such as urokinase-type plasminogen activator, convert inactive hepatocyte growth factor to its active form. The cytokines and the growth factors act in concert to initiate the reentry of quiescent hepatocytes (in the G₀ phase) into the cell cycle from the G₁ phase to the S phase, resulting in DNA synthesis and hepatocyte proliferation. The metabolic burden is indicated by the accumulation of bile acids in the blood, which enter the hepatocytes and drive increased protein and DNA synthesis. To signal the end of proliferation, transforming growth factor-β blocks further replication.

(From Clavien P-A, et al, 2007: Strategies for safer liver surgery and partial liver transplantation. N Engl J Med 356:1545-1559. Reprinted with permission.)

Immediately after liver resection, the rate of DNA synthesis in hepatocytes begins to increase, as they exit the resting state of the cell cycle (G₀) and enter G₁, traverse to DNA synthesis (S phase), and ultimately undergo mitosis (M phase). The induction of DNA synthesis occurs later in the nonparenchymal cells, at about 48 hours for Kupffer and biliary epithelial cells and at about 96 hours for endothelial cells (Grisham, 1969; Widmann & Fahimi, 1975). The first peak of DNA synthesis occurs at 40 hours after resection in rodents and at 7 to 10 days in primates. In small animals, the regenerative response returns the liver to the preresection mass in 1 week to 10 days. Clinical studies suggest that significant regeneration in humans is evident within 2 weeks after resection and is nearly complete at 3 months (Nagasue et al, 1987; Yamanaka, 1993). Studies in living donors, however, suggest that many donors do not reach 100% of starting liver volume by 3 months after donating their right lobe, and some are even lacking at one year (Pomfret et al, 2003; Nadalin et al, 2004; Ibrahim et al, 2005).

On a cellular level, hepatocyte proliferation starts in the periportal areas of the lobules (Rabes et al, 1976) and proceeds to the pericentral areas by 36 to 48 hours. Liver histology at day 3 to 4 after PHx is characterized by clumps of small hepatocytes surrounding capillaries, which change into true hepatic sinusoids. The hepatic matrix composition also changes from high laminin content to primarily containing fibronectin and collagen types I and IV. After a 70% hepatectomy, restoration of the original number of hepatocytes theoretically requires 1.66 proliferative cycles per residual hepatocyte. In fact, most of the hepatocytes in the residual lobes (95% in young and 75% in very old rats) participate in one or two proliferative events (Stocker & Heine, 1971). However, hepatocytes have an almost unlimited capacity to regenerate, and transplantation of several hundred healthy hepatocytes can repopulate a whole, damaged liver in a calculated minimum of 69 doublings (Rhim et al, 1994).

Liver Stem Cells

In contrast to other regenerating tissues, such as bone marrow and skin, primary liver regeneration after surgical trauma is not dependent on a small group of progenitor cells (stem cells). However, in some circumstances, such as in response to liver damage inflicted by agents such as galactosamine, hepatocytes are unable to replicate.

Induction of Proliferation: Priming and Cell-Cycle Progression

Within minutes after PHx, specific immediate early genes are activated in remnant hepatocytes (Haber et al, 1993). These genes include protooncogenes, which play an important role in normal cell-cycle progression—such as in JUN, FOS, MYC, and KRAS (Thompson et al, 1986; Morello et al, 1990; Taub, 2004)—and the transcriptional factors nuclear factor (NF) κB, signal transducer and activator of transcription 3 (STAT3), activator protein-1 (AP-1), and CCAAT enhancer–binding protein β (CEBPβ) (Cressman et al, 1995; FitzGerald et al, 1995). Historically, the onset of liver regeneration has been attributed to a flow-dependent response, by which increased relative flow after PHx resulted in hepatocyte proliferation and hyperplasia (Starzl et al, 1977). Experiments in parasymbiotic rats demonstrated the existence of humoral factors in the induction of liver growth after PHx (Moolten & Bucher, 1967). Interleukin (IL)-6 and tumor necrosis factor (TNF)-α have since been identified as the earliest factors triggering activation of several transcription factors during regeneration (Fig. 5.3) (Cressman et al, 1996; Yamada et al, 1997). In IL-6–deficient or TNF-α receptor–deficient mice, liver regeneration after hepatectomy is delayed (Streetz et al, 2000) but not completely abolished (Michalopoulos et al, 1984; Fujita et al, 2001). Therefore, other blood-derived mitogens, such as hepatocyte growth factor (HGF), were identified as a putative hepatic growth factor during liver regeneration (Fausto, 2000). Hepatocytes in normal liver will not respond to mitogenic signals without a set of “priming” events that switch them into a responsive state. This has been described by Webber and colleagues (1994), who identified priming factors, involved in initiating and triggering hepatic response to injury, and concomitant growth factors and their receptors, which allow competent hepatocytes to progress through the cell cycle. Priming is accomplished by the release of preformed cytokines that subsequently activate transcription factor complexes and allow the cell to exit G₀ into G₁ in the cell cycle. This group includes TNF-α and IL-6 (Aldeguer et al, 2002). Growth factors include the potent hepatocyte mitogens HGF, transforming growth factor (TGF)-α, and heparin-binding epidermal growth factor (HB-EGF). This process is further controlled by comitogens—such as insulin, glucagon, steroid and thyroid hormones, and epinephrine—that facilitate activity of the mitogens and by downregulation of growth factor inhibitors, such as activin A and TGF-β.

FIGURE 5.3 Stimulated by components of the innate immune system, Kupffer cells produce and secrete interleukin (IL)-6 and tumor necrosis factor-α (TNFα) to kick-start the regenerative response. IL-6 helps stimulate hepatocyte proliferation via transcription 3 (STAT3) activation; in turn, this response is negatively regulated by cytokine signaling-3 (SOCS3).

(From Alison et al, 2009: Stem cells in liver regeneration, fibrosis and cancer: the good, the bad and the ugly. J Pathol 217:282-298. Reprinted with permission of John Wiley & Sons.)

So far, few factors have been identified as possibly responsible for the release of these priming cytokines and growth factors in the onset of liver regeneration. The first is endotoxin lipopoly-saccharide (LPS), produced in the gut by gram-negative bacteria. Circulating LPS appears to be a strong signal for Kupffer cells to start the cascade that results in hepatocyte replication. Rats treated with antibiotics and germ-free rodents have a delayed peak of DNA replication after PHx, confirming the importance of LPS (Cornell et al, 1990). Another major finding is the demonstration that cytokine activation and DNA replication are severely impaired in mice lacking the complement components C3a and C5a (Strey et al, 2003). In particular, mice lacking both C3a and C5a have impaired production of TNF-α and IL-6 after PHx and poor activation of NFκB and STAT3. In the initiation of growth factors, urokinase-type plasminogen activator (uPA) appears to play an important role; uPA and its downstream effector, plasminogen, increase within 1 to 5 minutes after PHx and rapidly cleave the HGF precursor, pro-HGF. Blocking uPA delays the appearance of HGF and thereby delays liver regeneration, whereas blocking plasminogen-activator inhibitor (PAI) accelerates the release of HGF and accelerates liver regeneration (Currier et al, 2003).

More recently, new important factors in the initiation of liver regeneration have been identified. Extracellular adenosine triphosphate (ATP) has emerged as a rapid-acting signaling molecule that leads to quick and transient activation of JNK signaling after PHx, induction of immediate early genes c-FOS and c-JUN, and activator protein 1 (AP-1) DNA-binding activity (Thevananther et al, 2004; Gonzales et al, 2009). Also recently, an important role for blood platelets in I/R injury and after partial liver resections has been demonstrated. Thrombopenic animals exhibited reduced hepatocyte proliferation 48 hours after the initial ischemic insult, with diminished expression of TNF-α and IL-6 as early as 4 hours after reperfusion (Nocito et al, 2007). In a model of partial hepatectomy, the effect was shown to be mediated by serotonin, because mice lacking platelet serotonin displayed a lack of liver regeneration (Lesurtel et al, 2006). About 95% of all serotonin found in blood is stored in platelets and in vitro, and serotonin is a potent mitogen that stimulates hepatocyte mitosis (Balasubramanian & Paulose, 1998). The platelet effect on liver regeneration after PHx was mediated through the 5-HT2A and 2B receptor subtypes on hepatocytes and could be restored by injection with the serotonin precursor 5-HTP. These results suggest that a molecular action of serotonin is involved in the induction of hepatoctye proliferation after a major loss of hepatic tissue and that it may involve the release of growth factors, such as IL-6 (Clavien, 2008).

Intracellular Pathways and New Profiling Techniques

The importance of liver regeneration in sustaining life is characterized by the plethora of pathways involved and the fact that liver regeneration is usually only blunted or delayed in experimental models that lack a “critical” pathway. There are nonetheless several well-described patterns in liver regeneration. The immediate early genes that are activated in regenerating liver were first identified by Mohn and colleagues (1990) and Haber and colleagues (1993), who described induction patterns of more than 70 genes activated in the first week after two-thirds PHx in a rat model, genes normally not expressed in the quiescent liver. The development of cDNA microarray technologies set the stage for studying genome-wide expression and expanded this list to about 180 genes (Su et al, 2002; Arai et al, 2003).

A mouse cDNA array has also been used to study the crucial role of IL-6 in liver regeneration in IL-6–deficient mice after hepatectomy, showing a 36% reduction in the early gene expression compared with wild-type mice. In the absence of IL-6, activation of genes such as u-PA receptor (PLAUR) and SERPINE1, crucial for HGF activation and the MAPK pathway, were remarkably delayed (Li et al, 2002). IL-6 activates the JAK/STAT3 and MAPK signaling pathways via the gp130/IL-6R complex. This leads to activation of an array of immediate and delayed early genes required for normal liver-specific metabolic functions, repair, and protection from injury (Taub, 2003; Zimmers et al, 2003; Wuestefeld et al, 2003). STAT3 is crucial for cells to progress from the G₁ to S phase and for activating the MYC gene required for cell-cycle progression. Other intracellular signaling pathways that involve the receptor tyrosine kinases p38 MAPK, pERKs, and c-jun N-terminal kinase (JNK) are also rapidly activated.

Progression through the cell cycle is regulated by cyclins and cyclin-dependent kinases (CDKs). Various combinations bind to form kinase complexes that are active at distinct points within the cell cycle and tightly controlled by several mechanisms, including binding by CDK-inhibitory proteins, such as p21. Feedback signals to this process are provided by the suppressor of cytokine signaling-3 (SOCS3) and TGF-β, also regulated by IL-6. Other studies confirmed the importance of NFκB (Arai et al, 2003; Fukuhara et al, 2003; Togo et al, 2004) and showed immediate upregulation of apoptotic genes (FAS and caspases) in livers that failed owing to excessive resection (Fig. 5.4) (Morita et al, 2002). The cytokine-induced form of nitric oxide synthase (iNOS) seems to plays an important role in scavenging oxygen radicals and offering protection from apoptosis caused by a proinflammatory response mediated through IL-6 and TNF-α (Rai et al, 1998).

FIGURE 5.4 Intracellular pathways of liver regeneration. Multiple intracellular signaling pathways are rapidly activated. Progression through the cell cycle is regulated by cyclins and cyclin-dependent kinases (CDKs) tightly controlled by several mechanisms, including binding by CDK-inhibitory proteins, such as p21. (From http://en.wikipedia.org/wiki/File:Signal_transduction_pathways.png.)

The HGF/c-Met pathway is important for sustaining DNA synthesis after injury and activates various downstream pathways that involve PI3K, ERK/MAPK, and AKT1 (Huh et al, 2004). This pathway cross talks with the wnt/β-catenin signaling pathway, which has come to the forefront in liver biology over the last several years (Monga, 2009). Increased levels of HGF results in β-catenin dissociation along with nuclear translocation (Monga, 2002) and upregulation of downstream targets of this pathway, such as cyclin D1, MYC, PLAUR, matrix metalloproteinases (MMPs), and epidermal growth factor receptor (Michalopoulos & DeFrances, 1997). Vascular endothelial growth factor (VEGF) interacts with endothelial cells in the liver to increase HGF production from nonparenchymal cells (LeCouter et al, 2003).

In the transplant setting, microarray analysis of SFS rat liver grafts showed upregulation of vasoconstrictive and adhesion molecule genes at early time points postreperfusion, with later increases in genes associated with inflammation and cell death and downregulation of genes related to energy metabolism (Man et al, 2003), which was recently confirmed in the situation of clinical deceased-donor and living-donor liver transplantation (de Jonge et al, 2009).

Remodeling of the Liver

Remodeling of the newly regenerated liver tissue begins with the repopulation and maturation of nonparenchymal cells, such as endothelial cells, stellate cells, and biliary epithelial cells. Newly formed hepatocytes form clusters into which replicating endothelial cells invade to form new sinusoids. To restore normal architecture, stellate cells, located between endothelial cells and hepatocytes, synthesize extracellular matrix proteins and TGF-β1, which can regulate the production of hepatic extracellular matrix (see Chapter 6). VEGF, angiopoietins 1 and 2, TGF-α, fibroblast growth factors (FGFs) 1 and 2, and HGF all are likely involved in the angiogenic process. Angiostatin, an inhibitor of angiogenesis, causes delayed and suppressed liver regeneration in mice (Drixler et al, 2002; Vogten et al, 2004). Remodeling of the extracellular matrix is associated with the activation of the urokinase-plasminogen pathway and the membrane-bound matrix metalloproteinase (MMP) pathway. MMP-9 is one of the most active proteins during liver remodeling and regeneration (Kim et al, 2000). The MMPs—in combination with HGF, EGF, and TGF-β1—act to remodel the extracellular matrix, changing the levels of several extracellular matrix proteins such as collagen, fibronectin, laminin, and entactin. The maturation and thickening of the extracellular matrix seems to have an inhibitory effect on proliferating hepatocytes, potentially signaling the end of rapid regeneration (Kim et al, 1998).

Maintaining Liver Function During Regeneration

After volume loss, hepatocytes must adapt rapidly and seek a compromise between maintenance of continued differentiated function and cellular replication to permit survival. After toxic injury, resection, or transplantation, the balance is dramatically shifted to the crucial tasks of recovery and regeneration at the expense of normal hepatic metabolism. The success of restoring lost liver mass, repairing tissue injury, and resolving inflammation determines the ability of the liver to support normal metabolic function and determines its ability to recover (Fig. 5.5) (Strey et al, 2004). Several of the expressed immediate early genes encode enzymes and proteins involved in regulating gluconeogenesis, a critical process after PHx to compensate for the lost glycogen content and to produce sufficient glucose for the whole organism (Haber et al, 1995; Rosa et al, 1992). Rapid and increased expression of genes involved in glucose homeostasis is seen after PHx in animal models. Most notably, these include PCK, glucose-6 phosphatase (G6Pase), and IGFBP1, controlled at the level of transcription by insulin (downregulation), glucagon and cAMP (upregulation), and glucocorticoids (upregulation) (Mohn et al, 1990; Diamond et al, 1993). Livers lacking IGFBP1 display abnormal liver regeneration (Leu et al, 2003).

FIGURE 5.5 Metabolic balance between regeneration and maintaining liver function. A, In times of relative quiescence, there is a balance within the liver of metabolic function and continuous liver cell replacement or restructuring as needed. B, In times of stress or after injury or resection, the need for metabolic function or regeneration and recovery is increased. If metabolic need is great as a result of conditions within the patient, energy balance within the liver may not be sufficient to regenerate sufficiently, and the liver may not recover. If the requirements for regeneration and repair are overwhelming, metabolic function may be decreased, affecting patient outcome.

Decreased expression of genes that oppose gluconeogenesis also occurs rapidly posthepatectomy. Insulin itself can be a potent growth factor mediated through the insulin receptor, and insulin and glucagon have long been established as important “gut-derived” growth factors (Starzl et al, 1975). Liver-specific transcription factors—hepatocyte nuclear factors (HNFs)—play an important role in determining the level of glucose production, fatty acid metabolism, and liver-specific secreted proteins. CAAT/enhancer binding protein (C/EBP) α regulates expression of genes involved in hepatic glucose and lipid homeostasis, has antiproliferative proprieties, and is downregulated during liver regeneration after hepatectomy (Mischoulon et al, 1992; Costa et al, 2003).

During early regeneration, the liver accumulates fat. Neither the mechanisms responsible for nor the functional significance of this transient steatosis has been determined. Suppression of hepatocellular fat accumulation is associated with impaired hepatocellular proliferation after PHx, indicating that hepatocellular fat accumulation is specifically regulated during, and may be essential for, normal liver regeneration (Shteyer et al, 2004). Some data show that decreasing lipid peroxidation levels by vitamin treatment after PHx produces an attenuation of cellular apoptosis and a marked increase in the proliferation process, suggesting that the modulation of lipid peroxidation has a role in the liver regeneration process (Ronco et al, 2002). Hepatocytes in the periportal regions that divide and replicate after PHx require mitochondrial fatty acid β-oxidation. Peroxisome proliferator-activated receptor α, PPARα, may be a crucial modulator, controlling energy flux important for repair of liver damage and regeneration (Anderson et al, 2002). Expression of many other liver-specific genes, such as IGFBP1 and G6Pase, is regulated in the basal state by hepatic nuclear factor-1 (HNF1A). The transcriptional activity of HNF1A is upregulated during liver regeneration by the binding of HNF1A to the growth-induced transcription factors STAT3 and AP-1 (Leu et al, 2001). Together, growth-induced and tissue-specific transcription factors enable the liver to maintain metabolic function, despite the loss of two thirds of its functional mass.

In a microarray analysis of gene expression profiles after living-donor liver transplantation, it was recently demonstrated that C/EBPα was immediately downregulated, as was as HNF4α and PPARα (de Jonge et al, 2009). The immediate, marked downregulation of genes associated with pathways of lipid, fatty acid, and carbohydrate metabolism demonstrate how a liver segment needs to balance regenerating efforts with metabolic needs. It is this balance that limits the size of liver segment that can be transplanted or the size of remnant liver after donation or resection.

Termination of Proliferation

The size of the liver is highly regulated and is controlled by the functional needs of the organism. The most well-known antiproliferative factors within the liver are TGF-β and related family members such as activin (Derynck & Zhang, 2003). TGF-β is produced mainly by hepatic stellate cells, but in the early phase, it forms inhibitory complexes with SNoN and SKI proteins (Macías-Silva et al, 2002), rendering hepatocytes initially resistant to TGF-β (Koniaris et al, 2003). Later on, TGF-β acts through a heteromeric receptor complex, which subsequently phosphorylates proteins of the SMAD family, particularly SMAD2 and SMAD3 (Lonn et al, 2009). The SMAD2/SMAD3-SMAD4 complex translocates into the nucleus and activates target genes that negatively regulate the cell cycle (Nakao et al, 1997). Reactive oxygen species (ROS) enhance synthesis and activation of TGF-β (Koli et al, 2008), which may account for the reduced regeneration after ischemia and reperfusion. Interacting with the TGF-β/SMAD signaling could restore regeneration in a model of SFS liver grafts (Zhong et al, 2010).

Similarly, activin A blocks hepatocyte mitogenesis and shows diminished signaling during liver regeneration when its cellular-receptor level is reduced; its receptor level is restored once liver regeneration is terminated (Date et al, 2000). The level of activin-receptor mRNA expression was shown to be an important determinant in the magnitude of regeneration in portal vein ligation and PHx (Takamura et al, 2005a, 2005b). Suppressors of cytokine signaling (SOCS) are important negative regulators of cytokine signaling that prevent the tyrosine phosphorylation of STAT proteins. It has been shown that IL-6 signaling in the liver causes rapid upregulation of SOCS3, which correlates with the subsequent downregulation of phosphorylated STAT3, thereby terminating the IL-6 signal (Campbell et al, 2001).

Recent work strongly implicates the detection of blood bile acid levels by nuclear receptors as a regulator of liver growth (Huang et al, 2006). In addition, mammalian genes comparable to those in the Drosophila Hippo kinase-signaling cascade, which regulates wing mass during development, can also control hepatocyte proliferation (Dong et al, 2007). Overexpression of YES1-associated protein (YAP), the mammalian counterpart to yorkie (yki)—the last gene in the Drosophila Hippo kinase cascade—in a transgenic mouse model leads to massive liver hyperplasia, reaching 25% of body weight. YYA1P1 transcriptionally activates cell-cycle proteins, such as Ki-67 and c-Myc, and also activates inhibitors of apoptosis, and its phosphorylation by way of activation of the Hippo pathway blocks its ability to shuttle to the nucleus (Reddy & Irvine, 2008). It is therefore possible that the Hippo kinase pathway has a decisive role in determining overall liver size (Duncan et al, 2009).

Mechanisms of Liver Atrophy

The cause of liver cell death in atrophy is generally divided between necrosis and apoptosis. The distinction is important, because necrosis is a nonregulated traumatic disruption of a cell that occurs when it encounters overwhelming injury, whereas apoptosis is an inducible, highly orchestrated cascade of physiologic events. Necrotic cells lose membrane integrity, leak lysosomal enzymes, and induce a substantial inflammatory response. Apoptosis is energy dependent and allows cells to shrink and die without inducing inflammation. Apoptotic cells are characterized by plasma membrane blebbing, chromosomal condensation, and nuclear DNA fragmentation (Steller, 1995).

Portal Vein–Induced Hepatic Atrophy

The most common clinical scenario of hepatic atrophy is portal vein–induced atrophy, wherein occlusion of the portal vein leads to ischemia in areas of the liver; atrophy in this setting is a result of liver cell death secondary to necrosis and apoptosis (Ikeda et al, 1995; Shibayama et al, 1991). Ischemic necrosis of centrilobular areas of the liver predominates in the first 3 days of cell death. Areas peripheral to the necrotic liver cells predominantly undergo apoptotic cell death, and apoptosis persists long after necrosis subsides. Oxygen levels and mitochondrial function help determine which cells will undergo necrosis or apoptosis (Nishimura et al, 1998). Models of portal vein ischemia in rats have confirmed a caspase-dependent apoptosis and have indicated that Kupffer cells are involved in generating reactive oxygen substrates and other acute-phase reactants, culminating in mitochondrial dysfunction and apoptosis (Kohli et al, 1999).

Biliary-Induced Hepatic Atrophy

The molecular mechanisms involved in biliary obstruction leading to hepatic atrophy are much more centered on apoptosis, with little or no involvement of acute necrosis. Cholestasis results in the accumulation of toxic bile salts, which induce apoptosis through the Fas-mediated pathway. In this case, TNF-α and Fas ligand bind to the Fas death receptors, leading to a cascade of intracellular events, including cytochrome-c release from mitochondria and activation of apoptosis-mediating caspases. In Fas-deficient mice, bile duct ligation resulted in impaired apoptosis and less injury and fibrosis compared with wild-type mice (Canbay et al, 2002; Gujral et al, 2004; Miyoshi et al, 1999). More recent data, however, suggest a nonischemic model of necrosis/oncosis as the predominant process leading to cell death after common bile duct ligation, with cell swelling and without apoptotic caspase 3 activation (Fickert et al, 2005).

Clinical Causes of Atrophy

Hilar cholangiocarcinoma is a frequent cause of biliary atrophy, induced by occlusion of a major bile duct. Biliary occlusion, often accompanied by portal vein compromise, leads to atrophy of the liver approximately 20% of the time with this disease and has significant surgical implications. Also, postcholecystectomy bile duct strictures lead to hepatic atrophy 10% to 15% of the time (Hadjis & Blumgart, 1987; Pottakkat et al, 2009). Choledocholithiasis and hepatolithiasis are infrequent causes of atrophy, as are benign tumors such as papillomas, cystadenomas, and granular cell tumors (Blumgart et al, 1984; Blumgart & Kelley, 1984). Strictures caused by parasitic biliary infections, such as by Clonorchis sinensis and Ascaris lumbricoides, also have been known to cause biliary obstruction and associated atrophy of the liver. Occlusion of the hepatic artery alone would not induce atrophy.

Compensatory Regeneration Triggered by Atrophy

In the embolized portion of the liver, Kupffer cells release TNF-α and induce hepatocyte swelling and hyperplasia in the contralateral liver. Additionally, increased portal flow in the nonembolized lobe induces proliferation and activates several cytoplasmic growth-promoting signal-transduction pathways involved in hepatic regeneration, including JUN and the mitogen-activated protein kinase pathways. Kupffer cells in the contralateral liver begin to produce TNF-α as well, stimulated either by increased portal flow or through the direct effect of circulating growth factors (Kim et al, 2001). Nonparenchymal cells in the injured portion of the liver produce HGF. Messenger RNA levels of HGF in the ischemic portion of liver were noted to be 10 to 20 times that of normal, with most upregulation occurring in Kupffer cells (Hamanoue et al, 1992).

Patient-Related Factors

Intrinsic Liver Disease

The regenerative response of the liver can be seriously affected by preexisting intrinsic liver conditions such as steatohepatitis, fibrosis, and cirrhosis. Hepatic steatosis affects regeneration on several molecular levels. Lipid accumulation has been associated with hepatocyte mitochondrial damage caused by free-radical injury. Steatotic livers in rats show delayed mitosis and increased mortality after PHx, which may be due to abnormal TNF-α and IL-6 signaling (Selzner et al, 2000). The coordinated induction of Jnks and Erks is disrupted after partial hepatectomy in fatty livers of ob/ob mice (a model for steatohepatitis), with enhanced AKT and inhibition of PEPCK (Yang et al, 2001). Cyclin D1 induction is abolished along with Stat3 and reduced ATP levels, which may arrest cell-cycle progression. Hepatocyte mitochondrial damage associated with lipid accumulation is caused by free-radical injury from fatty acid oxidation. Abnormalities in induction of cytochrome P-450 may be one mechanism in the pathophysiology of these findings in fatty livers and may contribute to poor regeneration (Farrell, 1999; Kurumiya et al, 2000; Neuschwander-Tetri & Caldwell, 2003).

In clinical practice, the presence of underlying steatosis has considerable impact on operative morbidity and mortality after major hepatic resection (Behrns et al, 1998), with a significantly higher rate of complications if marked steatosis (≥30%) is present (see Chapters 65 and 87; Kooby et al, 2003). Steatohepatitis, or acute inflammation in the setting of fatty infiltration, carries an even higher risk and eventually results in fibrosis and cirrhosis. In transplantation, liver grafts with severe steatosis (≥60%) have higher rates of primary nonfunction, higher transaminases, and poorer graft survival in the transplant setting (Verran et al, 2003), possibly as a result of the inability to initiate repair and regeneration mechanisms. Progressive scarring and hepatic fibrosis lead to accumulation of abnormal collagen secreted into the extracellular matrix by stellate cells (Kim et al, 1998). Impaired regeneration is thought to be the result of the decreased diffusion of nutrients and hepatotrophic factors to the hepatocytes, and ultimately the architectural liver abnormalities created by scar contracture form a physical barrier that prevents hepatocytes from proliferating (Poynard et al, 1997).

Pharmacologic Therapy

Many exogenous agents can affect liver regeneration, including frequently prescribed drugs and neoadjuvant chemotherapy. Numerous medications are associated with impaired liver regeneration secondary to the induction of steatosis, including certain antiarrhythmic agents, antibiotics, antiviral agents, anticonvulsants, steroids, calcium channel blockers, statins, and antiglycemic medications. β-Blockers and nonsteroidal antiinflammatory drugs (NSAIDs) exert a more direct negative influence on liver regeneration. Liver regeneration after PHx is delayed in rodents treated with β-blockers and NSAIDs (Hong et al, 1997; Rudnick et al, 2001). β-Blockers may impair liver regeneration by decreasing portal blood flow to the liver and blocking the trophic effects of epinephrine. NSAIDs directly inhibit cyclooxygenase, part of the C/EBPA-mediated liver regenerative pathway. Although these effects have not been reflected in increased morbidity or mortality after liver resection in patients taking β-blockers or NSAIDs, the potential harmful effect of any medication on hepatic regeneration must be considered before resection.

In the past decade, more patients had liver surgery after neoadjuvant or induction chemotherapy. Major drawbacks of this hepatotoxic chemotherapy are the sinusoidal obstruction syndrome (SOS) associated with oxaliplatin (Rubbia-Brandt et al, 2004) and chemotherapy-associated steatohepatitis (CASH) (Vauthey et al, 2006), which is associated with irinotecan (see Chapters 65 and 87). Sinusoidal obstruction impairs liver regeneration after extensive liver resection and increases postoperative morbidity, but this may be prevented by administration of aspirin; CASH increases postoperative mortality—specifically, it increases deaths from postoperative liver failure (Fernandez et al, 2005).

Bevacizumab, a monoclonal antibody targeting vascular endothelial growth factor (VEGF) in combination with cytotoxic chemotherapy, appears to improve survival in patients with metastatic colorectal cancer (Hurwitz et al, 2004). Well-designed preclinical studies have demonstrated that inhibition of angiogenesis can inhibit wound healing (Scappaticci et al, 2005). Interestingly, no published studies have examined the effect of anti-VEGF therapy on human liver regeneration. Animal studies have demonstrated that liver regeneration depends on VEGF and angiogenesis (Drixler et al, 2002). In a murine model, anti-VEGF receptor therapy slightly impaired liver regeneration and cell proliferation after PHx compared with controls (Van Buren et al, 2008); however, no preclinical animal data on the effect of bevacizumab on liver regeneration has been shown, because it specifically recognizes human VEGF, not murine VEGF. Clinically no differences in postoperative liver insufficiency were seen if therapy was stopped at least 6 to 8 weeks before hepatic resection (Reddy et al, 2008; Zorzi et al, 2008).

Liver Transplantation

Regeneration is also crucial in liver transplantation. In deceased-donor transplantation, hepatocyte loss occurs in the form of I/R injury owing to the necessary preservation period from procurement to implantation and damage that may have occurred in the donor. Regenerative mechanisms are actively engaged after transplantation, depending on the length and degree of preservation injury (Debonera et al, 2001; Olthoff, 2002). Similarly, hepatocytes lost to the alloimmune response require replacement. Regeneration is also necessary when transplanting an SFS graft into a larger recipient. This is the sometimes the case in the setting of adult-to-adult living-donor transplantation (AALDLT) (Olthoff, 2003) and split-liver transplantation. Ischemic injury is minimized in AALDLT, because the preservation period is short; however, this technique supplies a graft that is by definition too small, requiring vigorous and immediate hepatocyte proliferation. By transplanting only 50% to 60% of what is the expected liver volume in adults, recipients (and donors) must rely on the rapid regeneration of a partial liver in addition to maintaining the basic metabolic functions required of the liver.

Warm and cold ischemic injury is an unavoidable component of transplantation. After prolonged cold ischemia of whole liver grafts, initiation of the cell-cycle pathways with upregulation of markers of liver regeneration occurs as described earlier. When ischemic injury is significant, a greater expression and activation of cytokines, transcription factors, and immediate early genes and a greater magnitude of hepatocellular replication is seen (Debonera et al, 2001). However, the liver can tolerate ischemic injury only to a certain “point of no return,” after which the damage is too extensive, and the liver or allograft cannot maintain functional homeostasis and regenerative capabilities, which results in liver dysfunction and graft failure (Debonera et al, 2002).

The amount of liver mass transplanted has been shown to be an important variable after transplantation. Early experimental studies addressing regeneration after transplantation showed that an SFS graft adapts to its environment and achieves a size equal to the original native liver (Kam et al, 1987). It became apparent that graft size–recipient ratio was crucial, as grafts that were too small had decreased survival (Francavilla et al, 1994). These findings correlated with clinical experience, in that SFS grafts regenerate to an appropriate size for the recipient; however, significant functional impairment of these grafts can occur, shown by prolonged cholestasis and histologic changes consistent with ischemic injury (Emond et al, 1996; Kiuchi et al, 1999; Lo et al, 1999; Zhong et al, 2006).

Using animal models of partial liver graft transplantation, the interplay between the regenerative response and ischemic injury in the setting of 50% and 30% size grafts was studied. The partial grafts showed a robust regenerative response if the ischemic injury was minimal. It became apparent, however, that when these partial grafts were subjected to ischemic injury for moderate to prolonged periods, a significant effect on survival was seen, with extensive hepatic necrosis, the inability to initiate or maintain the regenerative response, and decreased survival. These findings show the diminished tolerance of SFS grafts for additional injury beyond transplantation itself (Selzner et al, 2002; Debonera et al, 2004).

The amount of critical liver mass required for transplantation in living donation remains in question. Most centers have defined liver mass as graft-to-recipient body weight ratio (GRBWR) or as a percentage of the standard liver volume. No uniform method of measuring or reporting graft volume in relation to the recipient has been established. Clinical experience with living-donor and split grafts has led to an accepted lower limit of 0.8% GRBWR, or 40% of the standard liver volume (SLV), although significantly smaller grafts have been transplanted successfully. As mentioned before, donor liver and recipient characteristics may significantly influence these minimal accepted standard volumes. Patients with fulminant hepatic failure and those with significant metabolic stress may require more liver volume than stable patients transplanted under elective conditions (Marcos et al, 2000b).

Although it has been performed successfully, many centers are not performing AALDLT in acutely ill patients with fulminant failure because of the uncertainty of knowing whether a partial graft offers enough volume to support the recovery of such a recipient. As seen in experimental models, the accumulation of additional stressful stimuli, such as sepsis or renal failure, may push a relatively small graft into failure. Despite the actual measurement, if a small graft has any dysfunction, it can progress to what is termed small-for-size syndrome (SFSS). Within only 3 to 5 days after surgery, signs of liver failure—presence of two of the following on three consecutive days: bilirubin greater than 100 µmol/L, INR greater than 2, encephalopathy grade 3 or 4—with ascites, as well as renal and pulmonary failure, may become apparent (Dahm et al, 2005).

Stimulating Liver Regeneration Preoperatively

The assessment of hepatic function before resection is difficult, but we do know that the risk for perioperative complications increases when the remnant liver volume is too small, particularly in diseased livers (see Chapter 2; Yigitler et al, 2003). Hepatic function seems to recover quickly after resection but is difficult to measure. Conventional clinical blood tests and liver biopsy are inadequate measures of hepatic function. Child-Turcot-Pugh classification and the Model for End-Stage Liver Disease (MELD) score apply primarily to patients with cirrhosis and are only rough estimates of functional reserve. Bilirubin, albumin, international normalized ratio, and platelet count become abnormal only in advanced cirrhosis. Magnetic resonance imaging or computed tomography can measure residual liver volume accurately, but quantitative functional testing is not as precise. Traditional techniques to measure the volume of resection prior to hepatectomy can lead to inaccurate estimates of functional residual liver (FRL) volumes because of the presence of dilated bile ducts, multiple tumors, undetected lesions, compromised liver volume as a result of cholestasis or previous chemotherapy, cholangitis, vascular obstruction, steatosis or cirrhosis, or segmental atrophy and/or hypertrophy from tumor growth (Azoulay et al, 2000; Kubota et al, 1997).

In living-donor liver transplantation, volumetric imaging is used to determine total liver volume (TLV) and what the FRL volume will be following resection, and it can roughly be used to predict postresection function, as the donor liver is normal. In the recipient, the TLV of the diseased liver is not a useful index of function. Values calculated from GRBWR or standardized liver volume (SLV) based on recipient body surface area are used to predict minimum adequate graft volume (Vauthey et al, 2000; Higashiyama et al, 1993). A GRBWR greater than 0.8% or SLV greater than 35% are recommended to achieve successful graft and patient survival (Lo et al, 1999). For donor safety, the transplant community has recommended that at least 35% of TLV remain in the donor following resection for living donation. In comparison, extended resection of 80% of functional parenchyma can be performed in the absence of chronic liver disease for hepatobiliary malignancies (Abdalla et al, 2002). Recommended minimal FRL volume following extended hepatectomy is greater than 25% in a normal liver and greater than 40% in an injured liver, with moderate to severe steatosis, cholestasis, fibrosis, or cirrhosis, or following chemotherapy (Shoup et al, 2003).

Quantitative liver function tests measure the liver’s ability to metabolize or extract test compounds and can identify patients with impaired function at earlier stages of disease, but these tests have limited application in predicting a liver’s ability to regenerate after major resection (see Chapter 2). Clearance of indocyanine green is regarded as an accurate assessment of functional reserve that can help predict mortality (Lau et al, 1997), but more is being learned about measuring hepatic function in diseased livers using quantitative functional testing, such as methionine breath tests, cholate clearance, liver single-photon emission computed tomography scans, liver scintigraphy, and phosphorus 31 magnetic resonance spectroscopy (Corbin et al, 2002). When compared with indocyanine green (ICG) clearance and computed tomographic volumetric data, hepatobiliary scintigraphy is a reproducible and accurate tool to assess functional liver uptake and excretion, preoperative liver function reserve, and remnant liver function, and it allows monitoring of postoperative liver function regeneration (Dinant et al, 2007). Assessment of future remnant volume distinguishes those who will most likely benefit from preoperative liver enhancement techniques with PVE or hepatic artery embolization.

Therapeutic Use of Stem Cells

In addition to the classical first pathway (hepatocyte proliferation) and second pathway (hepatic progenitor cells) in liver regeneration, new evidence using bone marrow transplantation has begun to crystallize the existence of a third, extrahepatic pathway: the ability of bone marrow and cord-blood derived cells to engraft as hepatocytes (Friedman & Krause, 2009; Theise & Kraus, 2002).

Adult bone marrow includes two well-defined populations of stem cells: hematopoietic stem cells (HSCs), which give rise to all mature lineages of blood, and mesenchymal stem cells (MSCs), which can differentiate into bone, cartilage, muscle, and fat. Evidence for transition from bone marrow (BM) to hepatocyte has been demonstrated by analyzing liver samples after male-to-female BM transplantation in rodents and humans (Alison et al, 2000; Lagasse et al, 2000; Theise et al, 2000b). However, the extent to which this occurs and the mechanisms involved are still under debate (Fausto, 2004; Oertel & Shafritz, 2008). Estimates of repopulation by hematopoietic stem cells vary from 0.01% up to 40%, depending on the experimental conditions.

In the discussion about the mechanisms whereby the HSCs acquire a hepatocytic phenotype, both fusion of stem cells and hepatocytes (Wang et al, 2003b; Vassilopoulos et al, 2003) and transdifferentiation of stem cells into hepatocytes have been proven (Harris et al, 2004; Jang et al, 2004). The highest levels of BM-derived hepatocytes were found in humans with severe liver disease, suggesting that tissue damage may promote BM-derived cells engraftment as hepatocytes. Also, the release of adult stem cells from BM was shown after partial liver resection for benign and malignant conditions (Gehling et al, 2005; De Silvestro et al, 2004), and BM-derived stem cells increased the regeneration of contralateral liver after clinical PVE by 250% (Esch et al, 2005).

Recent studies report that MSCs promote liver repair in cases of liver damage (Alison et al, 2009). MSCs isolated from human umbilical cords (Yan et al, 2009), adipose tissue (Banas et al, 2008), bone marrow (Kuo et al, 2008), or rat bone marrow (Abdel Aziz et al, 2007) improved liver function in rodents suffering from acute liver damage (e.g., carbon tetrachloride injections). Infusion of hepatocyte-like cells, which were differentiated in vitro from MSCs, improved liver function following acute injury and appears to be therapeutically useful to animals with ongoing chronic hepatic damage (Fang et al, 2004; Zhao et al, 2005; Sakaida et al, 2004; Oyagi et al, 2006; Higashiyama et al, 2007). MSC therapy is sometimes considered to be a double-edged sword, and concerns were raised about MSCs promoting liver cirrhosis by differentiatng into myofibroblasts, which are involved in the fibrotic response (Russo et al, 2006; di Bonzo et al, 2008). The latest publications in this field indicate that the use of MSCs is safe and has a beneficial effect on liver fibrosis (Zhao et al, 2009; Chang et al, 2009; Roderfeld et al, 2009). Some phase I trials involving the injection of autologous BM cells into patients with cirrhosis have reported modest improvements in clinical scores (Lin et al, 2008; Houlihan & Newsome, 2008). Thus MSC-mediated liver therapy is a highly promising field; such therapy significantly improves liver function and increases animal survival in experimental liver-injury models; however, its role in liver regeneration after surgery remains to be determined.

Use of Antisense MicroRNAs

Recent studies have uncovered profound and unexpected roles for a family of tiny regulatory RNAs known as microRNAs (miRNAs) in the control of diverse aspects of hepatic function and dysfunction, including hepatocyte growth, stress response, metabolism, viral infection and proliferation, gene expression, and maintenance of hepatic phenotype (Elmen et al, 2008; Ambros, 2004). Some of these miRNAs are specific to the liver, and silencing of these resulted in increased expression of several hundred genes, including those normally repressed in hepatocytes. The significance of miRNAs in regenerating liver has not been established, but in a murine model, there was upregulation of some miRNAs with increasing age (Maes et al, 2008). In particular, three upregulated miRNAs may contribute to the aging-related decline in oxidative defense by targeting various classes of glutathione S–transferases. Other proteins in decline in old liver and targeted by the upregulated miRNAs are involved in mitochondrial functions or maintenance. Antagonizing these miRNAs may reverse the decline of regeneration and oxidative defense mechanisms in aging liver.