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From the Departments of Morphology and Molecular Pathology and Hepatology,* University of Leuven, Leuven, Belgium; and the Department of Medicine, The Johns Hopkins University, Baltimore, Maryland
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Abstract |
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Animal data indicate that oval cells are activated when oxidative stress inhibits the regenerative capacity of more mature hepatocytes.11,12 Oxidative stress is thought to play a major role in the pathogenesis of both alcoholic and nonalcoholic fatty liver disease (NAFLD).13,14 The replicative activity of mature hepatocytes is also known to be inhibited in patients with alcoholic hepatitis,15 rodents with alcohol-induced fatty livers,16 and in some animal models of NAFLD.17,18 Although the combination of oxidative liver damage and inhibited hepatocyte proliferation provides a strong stimulus for oval cell accumulation in the liver, whether or not this cell population is expanded in fatty liver disease (FLD) has, to our knowledge, never been studied.
We therefore studied three different murine models of FLD [ie, genetically obese (ob/ob) mice and normal mice with fatty livers induced either by ethanol (EtOH) or methionine choline-deficient (MCD) diets], as well as patients with FLD related to either NAFLD or chronic alcohol abuse.
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Materials and Methods |
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Animals
Adult (8 to 10 weeks of age) male C57BL-6 ob/ob mice, their lean littermates, and age- and gender-matched wild-type C57BL-6 were purchased from Jackson Laboratories (Bar Harbor, ME). Ob/ob mice and their lean littermates were fed standard chow. Wild-type mice were fed experimental diets: some (n = 32) received liquid diets that contained 4% EtOH (v/v) (BioServ Inc., Frenchtown, NJ) for 4 weeks; others (n = 32) were pair-fed the same volume of diet in which dextrin maltose was substituted isocalorically for EtOH; and still others were given methionine and choline-deficient diets (ICN diet catalog no. 96 0439, Costa Mesa, CA) (n = 24) or methionine choline-sufficient diets (n = 24) from the same manufacturer.
To induce hepatocyte proliferation, mice were subjected to 70% partial hepatectomy (PH) or treated with 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP gift from Dr. A. Columbono, University of Cagliari, Cagliari, Italy) or ethionine, pharmacological agents that induce hepatocarcinogenesis.19,20 PHs were performed under light ether anesthesia using the methods of Higgins and Andersen,21 as we described.22 Mice (n = 4/group/time point) were sacrificed either immediately before PH or at various times (0.5, 1, 24, 36, or 48 hours) after PH. PH was performed on 20 ob/ob mice, 20 of their lean littermates, and 45 wild-type mice that were fed either EtOH (Sigma, St. Louis, MO) (n = 25) or control (n = 20) diets. Twelve other ob/ob mice and 12 of their lean littermates were gavaged with a single dose of TCPOBOP (6 mg/100 g body weight) or an equivalent volume of vehicle (corn oil) and sacrificed 4 days later because this protocol induces significant hepatocyte DNA synthesis and hepatomegaly in normal mice.20 Finally, 24 chow-fed ob/ob mice and 24 mice that were fed either methionine choline-deficient diets (n = 12 ob/ob mice and 12 lean controls) or the respective control diet (n = 12 ob/ob mice and 12 lean mice) were given ethionine in their drinking water (final concentration, 0.15%) for 10 days to induce hepatic progenitor cell accumulation.23 In all of the aforementioned studies, one liver sample from each mouse was fixed in buffered formalin and another sample was snap-frozen in liquid nitrogen-cooled isopentane and stored at -80°C.
Immunohistochemistry
Four-µm-thick paraffin sections were deparaffinized and rehydrated, followed by heating in a microwave oven for 10 minutes at 750 W in citrate buffer, pH 6.0. Incubation with primary antibodies was performed at room temperature for 30 minutes. The polyclonal antibody against 56- and 64-kd human callus cytokeratins (dilution 1:200; DAKO, Glostrup, Denmark) was followed by undiluted anti-rabbit Envisiou (DAKO). Monoclonal mouse OV6 antibody (a kind gift from Stewart Sell, Albany Medical College, Albany, NY) was detected using the DAKO Animal Research Kit, peroxidase (DAKO). Oval cells (putative liver cell progenitors) were defined as small cells with an oval nucleus and little cytoplasm either singular or organized in arborizing ductular structures.24 Strong reactivity for cytokeratins and oval cell marker OV-6 were used for the assessment of progenitor cells. The interlobular bile duct was defined as being a tubular structure with a recognizable lumen and associated with a branch of the hepatic artery. Before each counting in a portal tract, the interlobular bile duct was looked for and excluded for the countings. The number of oval cells was subsequently assessed in each biopsy, by calculating the average number of hepatic oval cells per high-power field (HPF) based on a count of oval cells in five nonoverlapping HPFs, using a x40 objective.
Determination of Hepatic Mitochondrial Generation of Reactive Oxygen Species (ROS)
Mitochondria were isolated from fresh liver samples obtained at the time of sacrifice.7 Protein concentration was determined with the BSA assay (BioRad, Rockford, IL). Lucigenin (5 µmol/L bis N-methylacridinium; Sigma, Rockport, IL) was used to detect superoxide anion (O2-) and luminol (10 µmol/L 5-amino-2,3-dihydro-1,4-phthalazinedione, Sigma) was used to measure hydrogen peroxide (H2O2) generation. Briefly, mitochondria from an individual mouse were suspended in air-saturated reaction buffer (70 mmol/L sucrose, 220 mmol/L mannitol, 2 mmol/L Hepes, 2.5 mµmol/L KH2PO4, 2.5 mmol/L MgCl2, 0.5 mmol/L ethylenediaminetetraacetic acid, and 0.1% bovine serum albumin, pH 7.4) plus substrate (6 mmol/L of succinate) (final concentration, 0.1 mg mitochondrial protein/ml) at 37°C. Each reaction was performed in triplicate. O2- and H2O2 production by mitochondrial suspensions from six to eight mice/treatment group were evaluated by monitoring chemiluminescence continuously throughout a 60-minute period in a Berthold Biolumat LB9595 luminometer.25
Evaluation of ROS Regulatory Enzymes
Mitochondrial preparations from the same mice were also assessed for the content of reduced glutathione (GSH) and the activities of several enzymes that regulate ROS production/detoxification. GSH content was assayed according to a modification of the protocol originally described by Hissin and Hilf26 using reagents from Calbiochem Co. (San Diego, CA). Mitochondrial GSH content was calculated using a concurrently run standard curve. Mitochondrial superoxide dismutase (MnSOD) activity was measured in mitochondrial pellets (1 mg) by a modification of the assay described by Flohe and Otting27 using Calbiochem reagents, including 5,6,6a,11b-tetrahydro-3,9,10-trihydroxybenzofluorene, which undergoes alkaline autoxidation in a MnSOD-catalyzed reaction that yields a chromophore that absorbs maximally at 525 nm. MnSOD activity was calculated using a concurrently run standard curve. Mitochondrial glutathione peroxidase (GPx) activity was measured using reagents from Calbiochem. Mitochondrial protein (50 µg) were suspended in 1 ml of buffer (50 mmol/L Tris-HCl, pH 7.6, 5 mmol/L ethylenediaminetetraacetic acid, 1 mmol/L GSH, 0.22 mmol/L NADPH, and 0.4 U of glutathione reductase) and tert-butyl hydroperoxide (final concentration, 0.22 mmol/L) was added to initiate NADPH consumption, which was monitored at 340 nm in a spectrophotometer. One nmol of NADPH consumed/min/ml = 1 mU/ml GPx activity.
Human Tissue Samples
Patient Populations
We retrospectively studied 45 liver biopsies obtained from the files from the Department of Pathology, University Hospital Leuven. Twenty patients were diagnosed with NAFLD on the basis of histology, a history of diabetes mellitus or obesity, no history of alcohol abuse, and no evidence of other causes of liver disease on serological testing. Twenty-five patients with alcoholic liver disease (ALD) were selected on the basis of histology, a clinical history of alcohol abuse, and no evidence of other causes of liver disease on serological testing. As a control group, archival normal liver tissue was obtained from three patients without histopathological, biochemical, or serological evidence of liver disease.
Histopathology Assessment and Immunohistochemistry
For assessment of histopathology and fibrosis, the sections were routinely stained with hematoxylin and eosin, Hall’s, Sirius Red, PAS after amylase digestion. The degree of fibrosis was scored according to the Brunt classification of fibrosis/cirrhosis.28 We performed immunohistochemistry on B5-fixed, paraffin-embedded material, using monoclonal antibodies against cytokeratin 7, a known marker for hepatic oval cells and intermediate hepatocyte-like cells (HepLCs).7 The number of progenitor cells was subsequently assessed in each biopsy, by calculating the average number of oval cells per HPF based on a count of oval cells in five nonoverlapping HPFs, using a x40 objective. We used the same approach as in the mice models: the interlobular bile ducts were located and excluded from the countings, while ductules and single progenitor cells were counted as the ductular progenitor cell compartment.
More extensive phenotyping of the oval cells and their progeny was performed as previously described,7 using antibodies against OV-6, cytokeratin (CK)-19, chromogranin-A (chrom-A), and neural cell adhesion marker on selected frozen biopsies of five NAFLD and five ALD patients. Cells were scored as oval cells or their progeny on the basis of morphological and immunohistochemical criteria.7,24,29
Electron Microscopy
Electron microscopy was performed on small liver fragments of four patients in the NAFLD group and six patients in the ALD group. The fragments were immediately fixed in 2.5% glutaraldehyde and 0.1 mol/L of phosphate buffer. After postfixation in 1% osmium tetroxide and 0.1 mol/L of phosphate buffer, the samples were dehydrated in graded series of alcohol and embedded in epoxy resin. Ultrathin sections were cut, stained with uranyl acetate and lead citrate, and examined using a Zeiss EM 10 electron microscope.
Statistical Analysis
Analysis of variance was used to compare differences in Ov-6 (+) cell numbers, ROS production, ROS-regulatory enzyme activities, and GSH content among the various groups of mice. The significance of the correlation between the oval cell/HepLC count and the extent of fibrosis was assessed using the Kruskal-Wallis test. The Wilcoxon-Mann Whitney test was used to determine whether the difference in grade of activation of oval cells and HepLCs between ALD and NAFLD was significant. Significance was accepted for P values <0.05.
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Results |
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Oval Cell Accumulation
In the absence of any added stimulus for liver regeneration, the numbers of oval cells were significantly increased in all three groups with fatty livers (Figure 1
, Table 1
).
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Various pharmacological agents that cause oxidative stress inhibit DNA synthesis in mature hepatocytes and induce compensatory mechanisms, including the recruitment of hepatic progenitors.19,23
In view of reports that FLD inhibits replication of mature hepatocytes17,18
and our new evidence for oval cell accumulation in hepatic steatosis, we postulated that oxidative stress would be a common feature of the various murine models of FLD. We therefore assessed the activities of various enzymes that regulate ROS generation, glutathione (GSH) content, and O2- and H2O2 production in fatty liver and control liver mitochondria. Compared to control mitochondria, fatty liver mitochondria uniformly demonstrated alterations in ROS-regulatory enzymes and/or GSH content that favor excessive ROS production (Table 2)
. For example, the activity of manganese superoxide dismutase (MnSOD), the enzyme that converts O2- to H2O2, is increased in all three groups with fatty livers. This is compounded by reduced activity of glutathione peroxidase (GPx) in ob/ob mitochondria and decreased mitochondrial GSH content in mitochondria from EtOH-fed mice. As predicted, mitochondrial ROS production is increased significantly in all three types of fatty liver, although the relative contributions of O2- and H2O2 to this process appear to vary among the different models (Table 3)
. For example, mitochondria from MCD diet-fed mice exhibit the greatest production of O2-, whereas those obtained from ob/ob mice produce the highest levels of H2O2. Compared to the other two groups with fatty livers, mitochondria from EtOH-fed mice produce intermediate levels of both O2- and H2O2. Whether or not these differences in the types of ROS that predominate in various models of FLD have biological significance is unknown. However, this merits consideration because greater numbers of oval cells were observed in EtOH-fed and ob/ob mice, which produced higher levels of H2O2 than MCD diet-fed mice, which produced relatively little H2O2 despite generating the highest levels of O2- (Figure 1
, Table 1
).
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Progenitor cell activation has mainly been reported in rat models associated with inhibited proliferation of mature hepatocytes. By monitoring hepatocyte expression of proliferating cell nuclear antigen and incorporation of BrdU, we recently showed that replication of mature hepatocytes is severely impaired after PH in mice with obesity-related18
or alcohol-induced30
FLD, compared to their respective healthy controls. Here, we studied liver samples from these same mice to determine whether inhibited hepatocyte replication was accompanied by the hepatic accumulation of oval cells after PH. As shown in Figure 2a
, slight variations in the numbers of oval cells occur in normal mice after PH, with a small (but highly significant, P < 0.0005) increase in oval cell accumulation occurring at 
36 hours, the time period when hepatocyte DNA synthesis typically becomes maximal in healthy livers. Before PH, significantly greater numbers of Ov-6 (+) cells are demonstrated in both fatty liver models (ie, EtOH-fed and ob/ob mice) (P < 0.0001 for mice with fatty livers versus control mice at time 0). In addition, wide fluctuations in the size of the putative progenitor cell population occur after PH. By 36 hours after PH the numbers of oval cells in the fatty liver remnants have increased by 70%, leading to a significant increase in the size of this cellular population compared to pre-PH values (P < 0.01 at 36 hours compared to time 0). Moreover, by 36 to 48 hours after PH, in addition to single oval cells and strands of oval cells, intermediate hepatocyte-like cells, in continuity with oval cells, are abundant in the fatty livers, but not in controls. These intermediate hepatocyte-like cells are larger and show a submembranous cytokeratin-staining pattern, instead of the cytoplasmic staining pattern of small oval cells (Figure 2, b and c)
. This finding suggests that oval cell differentiation may contribute to the regeneration of fatty livers after PH.
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. Compared to PH (which does not cause hepatocellular carcinoma in normal mice), TCPOBOP treatment was followed by significantly greater accumulation of oval cells in both lean and ob/ob mice. As noted after PH, after TCPOBOP treatment, the numbers of oval cells in mice with fatty livers were significantly greater than controls (Table 1)
. Thus, in ob/ob mice that exhibit chronic oxidative stress and inhibited replication of mature hepatocytes after either PH or toxic liver injury, the ensuing regenerative responses are characterized by increased hepatic accumulation of putative progenitor cells.
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As others have shown, ethionine expanded the oval cell population in lean control mice that were fed antioxidant-depleted diets (Table 1)
. However, the drug dramatically increased the hepatic accumulation of Ov-6 (+) cells in chow-fed ob/ob mice. In fact, the numbers of Ov-6 (+) cells in the livers of ethionine treated, chow-fed ob/ob mice significantly exceeded the numbers of these cells in the livers of one-half MCD plus ethionine-treated wild-type mice (Table 1
and Figure 3, a and b
).
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Immunohistochemistry
Progenitor cells resembling oval cells were rarely detected in the normal controls. However, in patients with NAFLD and in those with ALD, we consistently noted small single oval cells (CK7, CK19, Ov-6, chromogranin-A+ cells). These cells were located in the periportal area, and often extended into the parenchyma of the lobule. Reactive ductules (CK7, CK19, Ov-6, chromogranin-A, neural cell adhesion marker + structures) were also seen at the portal-parenchymal interface. Because bile ductules harbor the progenitor cells in the canal of Hering, we considered the whole of reactive ductules and single oval cells the expanded progenitor cell compartment. Intermediate hepatocyte-like cells (HepLCs) that express CK7, chromogranin-A, and OV-6, were seen in continuity with the oval cells (Figure 4)
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. Thus, putative hepatic progenitor cells accumulate in the livers of patients with FLD. Also, as we have demonstrated in patients with chronic viral hepatitis,9
in both ALD and NAFLD patients, the numbers of putative hepatic progenitors increases with the severity of the underlying liver disease.31
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The overall ultrastructural features of the liver parenchyma of NAFLD patients and ALD patients were similar and comparable to those described in the literature.32
Of note, mitochondrial variations in sizes and shape, as well as rearrangement of cristae and paracrystalline inclusions were seen in both the NAFLD and the ALD biopsies, and there were no significant differences in the extent of mitochondrial pathology between the two groups. In all liver biopsies of NAFLD patients, small- to intermediate-sized progenitor cells, with epithelial characteristics, were identified in the periportal area (Figure 5)
. These small cells were found immediately adjacent to, or wedged between, hepatocytes. The hepatocytes adjacent to the small- to intermediate-sized cells typically exhibited abnormal mitochondrial morphology. The small cells themselves had an oval or round small nucleus, and a round nucleolus was typically noted at the nuclear margin. The cytoplasm contained a full assortment of organelles, and several bundles of tonofilaments were always obvious. These small cells are clearly different from endothelial and Kupffer cells. Endothelial cells, lined along the sinusoidal wall, are flattened and elongated cells with thin cytoplasmic processes, provided with fenestrae arranged in so-called sieve plates. A characteristic of these cells is the presence of pinocytotic pits and vesicles interfacing with a complex system of vacuoles, coated vesicles, and tubules. No tonofilaments are present. Kupffer cells are rather large star-shaped cells anchoring to the endothelial wall and bulging into the sinusoidal lumen. They are provided with numerous irregular microvilli and pseudopodia; their peripheral cell membrane contains numerous pinocytotic, phagocytic, and worm-like structures. In their rather voluminous cytoplasm many heterozygous lysosomes of different sizes are apparent. No tonofilaments are seen. Therefore, ultrastructural criteria clearly distinguish the small epithelial progenitor cells from other types of liver cells. In addition, in our tissue samples a basement membrane surrounded the sinusoidal pole of the small cells and early or well-formed junctional complexes of the desmosomal type joined them with adjacent hepatocytes. More voluminous small epithelial cells with a round nucleus were also noted, often forming a hemicanaliculus with neighboring hepatocytes. Fully developed junctional complexes joined these cells to hepatocytes.
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Discussion |
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In addition, we show that ROS-related replicative senescence in mature hepatocytes is accompanied by expansion of oval cells (a putative liver progenitor cell population) in both mice and humans with FLD. Both in the murine models and in humans, no significant difference in the degree of progenitor cell activation was noted between NAFLD and ALD. This indicates that, similar to oxidant-induced replicative senescence, the progenitor cell response is somewhat stereotypical, ie, not related to a specific type of toxin or injury. These results are in good agreement with our previous findings of human progenitor cell activation across various degrees of chronic hepatitis, as well as in various etiologies of necrotizing hepatitis (toxic, viral, or autoimmune).9,7,35 Taken together, these observations support the concept that oval cell expansion is a component of the liver’s adaptive response to oxidative stress.
In humans, the degree of oval cell activation showed a positive correlation with the degree of fibrosis, a marker of liver disease chronicity (P = 0.0017). Moreover, the number of intermediate hepatocyte-like cells increased with the stage of the liver disease (P = 0.0077), suggesting cumulative hepatocyte loss promotes not only oval cell accumulation, but also the differentiation of oval cells toward hepatocytes. For example, the number of oval cells and HepLC was most impressive, both immunohistochemically and ultrastructurally, in the ALD patients who had progressed to cirrhosis. The ultrastructural appearance of the intermediate hepatocytes in cirrhotic liver was so strikingly normal (ie, undamaged) that this highly suggests that they originate from the putative progenitor cells. In noncirrhotic mice with obesity or alcohol-induced fatty livers, intermediate hepatocyte-like cells were recognizable at 36 and 48 hours after PH, and after ethionine treatment, consistent with the human evidence that a reduced mass of functional, mature hepatocytes (rather than cirrhosis per se) triggers the differentiation of oval cells toward hepatocytes. Interesting in this regard is our recent evidence that the expression of ATP-binding cassette proteins MRP-1, MRP-3, and MDR-1 is increased in the progenitor cell compartment in various human liver diseases.36 These transporters are known to be cytoprotective. For example, MRP-1 helps cells to secrete GSSG or the GSH conjugates of 4-hydroxynononeal, products of oxidative stress reactions.37 Therefore, the phenotype of oval cells may convey a survival advantage that permits them to accumulate in conditions that severely damage more mature hepatocytes. This, in turn, helps to explain the positive relationship between oval cell and HepLC accumulation and reductions in mature hepatocyte mass: oxidant stress reduces the viability and expansion of mature hepatocytes, while sparing less mature hepatic progenitors. A similar paradigm has been implicated during neoplastic transformation.3 Thus, it is tempting to speculate that hepatocellular carcinoma develops because chronic oxidative stress exerts a selection pressure that favors the outgrowth of progenitor cell clones that are most resistant to oxidant damage. Further studies are needed to evaluate this possibility. Fortunately, the present work proves that there are good small animal models for human FLD. These can be used to unravel the pathogenic mechanisms for FLD progression, including the evolution of hepatocellular carcinoma.
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Footnotes |
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Supported by research grants from the National Institutes of Health (RO1 AA010154 and RO1 DK3579 to A. M. D.) and F. W. O. Vlaanderen (G.0139.00N to T. R.).
Accepted for publication June 12, 2003.
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