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Insights into disease-specific mechanisms for liver repopulation are needed for cell therapy. To understand the efficacy of pro-oxidant hepatic perturbations in Wilson disease, we studied Long-Evans Cinnamon rats with copper toxicosis under several conditions. Hepatocytes from healthy Long-Evans Agouti rats were transplanted intrasplenically into the liver. A cure was defined as lowering of copper to below 250 micrograms per gram liver, presence of atp7b mRNA in the liver and improvement in liver histology. Treatment of animals with the hydrophobic bile salt, cholic acid, or liver radiation before cell transplantation produced cure rates of 14% and 33%, respectively; whereas liver radiation plus partial hepatectomy followed bv cell transplantation proved more effective with cure in 55%, p<0.01; and liver radiation plus cholic acid followed by cell transplantation was most effective, with cure in 75%, p<0.001. As a group, cell therapy cures in rats preconditioned with liver radiation plus cholic acid resulted in less hepatic copper indicating greater extent of liver repopulation. We observed increased hepatic catalase and superoxide dismutase activities in Long-Evans Cinnamon rats, suggesting chronic oxidative stress. After liver radiation and/or cholic acid, hepatic lipid peroxidation levels increased, indicating further oxidative injury., although we did not observe overt additional cytotoxicity. This contrasted with healthy animals where liver radiation and cholic acid produced hepatic steatosis and loss of injured hepatocytes. We concluded that pro-oxidant perturbations were uniquely effective for cell therapy in Wilson disease due to the nature of pre-existing hepatic damage.
The potential of liver cell therapy has been studied in several animal models, although new mechanisms are required for achieving therapeutic levels of liver repopulation (1). Recent studies in rat and mouse models demonstrated that coupling of hepatic genotoxic damage and oxidative stress in the native liver benefited liver repopulation with cells (2–6). Similarly, hepatotoxicity induced by toxic transgenes or other genetic mechanisms has been effective in liver repopulation with cells (7,8). Nonetheless, the responses of healthy and diseased native liver cells to conditioning regimens may be different, which may alter cell therapy outcomes (9,10). Therefore, insights into mechanisms of transplanted cell proliferation under disease-specific conditions are needed.
Wilson’s disease (WD) constitutes an appropriate condition for study and is amenable to cell and gene therapy (4,9–13). This condition arises from mutations in the copper-transporting gene, ATP7B, with retention of copper in the liver, brain and other organs (11). Excellent rat and mouse models of WD have been developed (14,15). In the Long-Evans Cinnamon (LEC) rat, cell transplantation after hepatic preconditioning with DNA adduct-forming alkaloid, retrorsine, produced liver repopulation with healthy hepatocytes (4), whereas without preconditioning, liver was either not repopulated or required much longer (9). Similarly, hepatic preconditioning with radiation (RT) and ischemia-reperfusion (IRP) proved less effective in LEC rats (10).
To develop further insights in mechanisms of hepatic conditioning for cell therapy in WD, we addressed the potential of hydrophobic bile salts, which produce liver injury in several ways, including oxidative stress (16–19). We were especially interested in defining whether hydrophobic bile salts will synergize with other forms of oxidative stress and promote proliferation of healthy hepatocytes in the liver. Here, we report cell transplantation studies in the LEC rat to define this conditioning mechanism in the context of WD.
LEC rats, syngeneic healthy LEA rats and F344 rats were bred in the Special Animal Core of Marion Bessin Liver Research Center. Rats studied were 8 to 10-week old and were housed in 14h light and 10h dark cycles with unlimited access to water and chow containing 11.8 mg copper/Kg (Ralston Purina, St. Louis, MO). Some rats were given chow with 0.1% cholic acid (Test Diets, Harlan Teklad, Madison, WI).
Animals were anesthetized with inhaled ethyl ether or ketamine and xylazine. The Animal Care and Use Committee at Albert Einstein College of Medicine approved animal protocols according to NIH guidelines.
Hepatocytes were isolated from LEA donor rats by two-step collagenase perfusion. Cell viability was analyzed by trypan blue dye exclusion. For transplantation, 1×107 viable cells were suspended in 0.5 ml RPMI 1640 medium and injected over 10 to 15 s in the splenic pulp within 2h after isolation. Hemostasis was secured with ligature around injection site at the lower pole of the spleen.
Anterior liver lobes were removed according to Higgins and Anderson method for as described previously (20).
Total oxidase activity was measured with 1 mg/ml dimethoxybenzidine dihydrochloride (o-dianisidine; all chemicals were from Sigma Chemical Co., St. Louis, MO) in 0.1 M sodium acetate buffer, pH 5.6. To 10 μl serum was added 20 μg o-dianisidine with sodium acetate buffer to 100 μl total volume. For negative controls, 0.1 mg sodium azide was added to an aliquot of each sample. After adding 100 μl of 9 M sulfuric acid, reactions were continued for 90 min at 37°C. Absorbance at 540 nm was compared with corresponding negative controls. Standard curves were obtained with ceruloplasmin and oxidase activity was converted to ceruloplasmin in milligram per deciliter.
Liver samples were desiccated at 65°C under vacuum for 12h. Bile was collected by cannulating bile duct with P20 tubing at intervals before and after intrasplenic injection of copper-histidine as described previously (21). Bile and liver samples were stored at −20°C. Tissues were solubilized in nitric acid and tissue and bile copper was measured by graphite furnace atomic absorption spectroscopy.
RNA was extracted from frozen samples with Trizol™ Reagent (Life Technologies, Grand Island, NY). Atp7b and β-actin mRNAs were co-amplified by RT-PCR with a commercial kit (Access RT-PCR, Promega Corp., Madison, WI). PCR primers and conditions were as described (4). PCR products were resolved in 1.8% agarose containing ethidium bromide. Expected PCR products for atp7b and β-actin were 380 and 200 base pairs, respectively.
Liver samples were homogenized in 5% sulfosalicylic acid (100 mg tissue per milliliter). Cell debris was removed under 10,000 g for 5 min at 4°C. Glutathione assay was performed as described previously (22). Absorbance of reaction product was measured at 412 nm over 2 min and data were plotted against curves using glutathione standards. All analyses were in triplicate. Total protein concentration in aliquots was measured by Bradford assay.
Liver was homogenized in phosphate buffer (200 mg tissue per milliliter of 260 mM monobasic potassium phosphate and 40 mM dibasic sodium phosphate dehydrate). Cell debris was removed by centrifugation at 10,000 g for 10 min at 4°C. Catalase activity was measured as described previously (22). Time (T) for change in optical density from 0.45 to 0.40 at 240 nm was measured at room temperature. Catalase activity was estimated from the formula, 17/T=units/assay mixture. Protein content was determined in aliquots by Bradford assay. All assays were performed in triplicate.
Cu/Zn-, Mn- and Fe-SOD was measured with a commercial assay (kit 706002; Cayman Chemical Company, Ann Arbor, MI). Tissue was homogenized (100 mg per milliliter of 20 mM HEPES, 1 mM EGTA, 210 mM mannitol and 70 mM sucrose, pH 7.2). Cell debris was pelleted under 1,500 g for 5 min at 4°C. SOD was assayed at room temperature according to manufacturer’s instructions. Absorbance of reaction product was measured at 450 nm and results plotted against SOD standards provided.
The assay measured ThioBarbituric Acid Reactive Substances (TBARS) (kit 10009055; Cayman). Livers were ultrasonicated at 40V for 15 s (100 mg liver per milliliter of RIPA buffer: 50 mM Tris HCl, 0.25% sodium deoxycholate, 1% NP-40, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mM sodium vanadate, 1 mM NaF, and 1 μg/ml each of aprotinin, leupeptin and pepstatin). Cell debris was removed under 1,600 g for 10 min at 4°C. Sample OD was measured at 530 nm and concentration of MDA-TBA adducts was obtained from standard curves.
Liver samples were fixed in 10% buffered formalin, embedded in paraffin and sections were stained with hematoxylin and eosin. Macro- and microvesicular steatosis, polyploidy, apoptosis and mitosis were graded as described previously (23). The maximal possible score with advanced liver damage was 13 and minimal possible score in healthy livers was 2.
Data are shown as means ± SD or as median and range, according to parametric or nonparametric distributions, respectively. Significances were analyzed by χ2 test with Yates correction, Student’s t-test and Kruskall-Wallis analysis of variance (ANOVA) with Dunn’s test for multiple pairwise comparisons of group ranks (SigmaStat 3.1; Jandel Scientific, San Ramon, CA). P<0.05 was considered significant.
We established groups of LEC rats, including those without perturbations (Group I), and rats subjected to manipulations (Groups II–V) (Fig. 1A). In group II, rats received hepatic RT alone followed 2 weeks later by cell transplantation. In group III, rats received hepatic RT plus two-third PH 2 weeks before cell transplantation. In group IV, rats received 1% cholic acid for 3 weeks with cell transplantation 2 weeks after commencing the diet. In group V, rats received hepatic RT and 1% cholic acid prior to cell transplantation. As indicated in the study time-line (Fig. 1B), outcomes were analyzed after 6 months.
A cure was defined as detectable hepatic atp7b mRNA, copper content <250 μg/g dry liver weight and improved liver histology. Additional groups of LEC rats and F344 rats were administered cholic acid or hepatic RT without cell transplantation to determine the effects of these manipulations. These animals were grouped as follows: untreated rats as controls; rats given RT alone; rats given 1% cholic acid diet for 3 weeks; and rats given RT and then cholic acid diet for 3 weeks. Animals were sacrificed one week after treatments for tissue analysis. Healthy LEA rats and F344 rats served as controls for assays.
The difference in the numbers of animals in various groups was due to variable availability of LEC rats. It should be noted that LEC rats are relatively difficult to breed. More animals were included in some groups to ensure availability of sufficient numbers for statistical comparisons. Also, we were restricted to RT-PCR for atp7b as an indicator of liver repopulation in LEC rats because reliable antibodies to rat atp7b protein are not available. Detection of atp7b mRNA by our assay required repopulation of >10% of the LEC rat liver with healthy transplanted cells (4).
In group I, without any manipulations, LEC rats showed significant liver injury with serum ceruloplasmin of 0 to 5 mg/dl, undetectable atp7b mRNA, mean hepatic copper of 958±168 μg/g, absence of bile copper excretion, and abnormal liver histology with extensive cholangiofibrosis, hepatic polyploidy, and interspersed mitotic activity or apoptosis. This agreed with anticipated liver injury in the setting of copper toxicosis due to WD.
We examined outcomes 6 months after cell transplantation, as this time-frame was appropriate for analyzing liver repopulation in LEC rats treated with hepatic RT in previous studies (10).
In groups II–V, cell therapy in several animals with hepatic preconditioning met our criteria for cure. After cell transplantation with hepatic RT alone in group II, 6 of 18 rats (33%) were cured, whereas in group III after preconditioning with RT plus PH and cell transplantation, 6 of 11 rats (55%) were cured (Fig. 2). In group IV, where cell transplantation followed hepatic conditioning with cholic acid alone, only 1 of 7 rats (14%) was cured. Finally, in group V, where RT plus cholic acid constituted hepatic preconditioning, 9 of 12 (75%) rats were cured, which was most effective, p<0.05, ANOVA - Dunn’s test.
Grouped data analysis indicated that LEC rats meeting our criteria for cure showed significant improvements (Table 1). Again, these parameters improved most in group V animals treated with RT plus cholic acid.
RT-PCR for hepatic atp7b mRNA as well as liver histology showing improvements were in agreement with other findings (Fig. 3). In particular, consistent with appearance of atp7b mRNA and decrease in hepatic copper content, liver histology improved after cell therapy in LEC rats with therapeutic cure. By contrast, untreated LEC rats in group I or LEC rats without therapeutic responses across various groups showed extensive liver injury (Fig. 3B–G).
The contrast in histology grading between untreated LEC rats in group I and animals in group V meeting the definition of cure after preconditioning with RT plus cholic acid is given in Table 2.
We considered that inter-group and even intra-group responses to cell therapy were due to differences in transplanted cell engraftment and/or proliferation under the experimental conditions, arising from perturbations in the hepatic microenvironment. It should be noteworthy that in healthy F344 rats subjected to cell transplantation after RT-based preconditioning, animal-to-animal variability in proliferation of transplanted cells was infrequent or not encountered (5). Therefore, we compared changes in F344 rats and LEC rats.
In the first instance, we examined responses of healthy F344 rats and LEC rats to conditioning with RT and/or cholic acid 4-weeks after these treatments without cell transplantation. Liver histology in untreated healthy F344 rats was normal, whereas LEC rats showed significant baseline morphological abnormalities, including hepatic polyploidy and nuclear changes, as before (Fig. 4). In F344 rats, morphological changes after RT or cholic acid alone were restricted to enlargement of hepatocyte nuclei. After RT plus cholic acid, F344 rats showed further alterations with fatty change and losses of hepatocytes, especially in perivenous areas. By contrast, in LEC rats, no additional morphological changes were observed after RT, cholic acid, or RT plus cholic acid, although occasional apoptotic hepatocytes were present.
Next, we examined oxidative stress by measuring hepatic catalase, total glutathione, SOD and malondialdehyde. Typically, depletion of catalase, glutathione or SOD indicates exposure of cells to acute oxidative stress, whereas increased malondialdehyde content represents lipid peroxidation due to acute or chronic oxidative stress (22). In comparison with healthy F344 rat livers, catalase and SOD activities increased in LEC rat livers by an average of 3- and 3.8-fold, respectively, p<0.001, t-tests; while total glutathione activity was 2.8-fold less, p<0.001, t-test; and malondialdehyde content was unchanged, p=n.s. (Fig. 5). This suggested significant differences in oxidative stress in the two situations.
Also, after exposure to RT, cholic acid, or RT plus cholic acid, we observed increases in hepatic catalase and SOD activities in F344 rats, as well as LEC rats, suggesting an appropriate directionality of the biochemical change. By contrast, total hepatic glutathione content decreased in F344 rats in response to all these treatments, whereas in LEC rats, exposure to RT increased hepatic glutathione levels, although cholic acid did not change hepatic glutathione levels. Finally, we observed divergent responses with respect to hepatic malondialdehyde levels, which increased in LEC rats after RT, cholic acid, or RT plus cholic acid, whereas malondialdehyde levels declined in all conditions in F344 rats.
These studies demonstrated significant differences in transplanted cell proliferation and liver repopulation in healthy liver versus liver with copper toxicosis, oxidative injury and other perturbations. The preconditioning regimens in our studies did not produce mortalities due to hepatic changes. Similarly, presence of acute or chronic liver damage does not prevent engraftment of transplanted cells in the liver, as indicated by cell transplantation studies in animals with toxin-induced acute liver failure or chronic liver fibrosis (24,25). However, as preconditioning regimens exerted different effects on the kinetics of transplanted cell proliferation in normal animals and animals with pre-existing liver damage, it should be appropriate to emphasize the role of disease-specific mechanisms in optimizing hepatic preconditioning for liver repopulation with cells. This consideration was particularly applicable to cell therapy following induction of additional oxidative stress in WD, where RT and cholic acid proved most effective, RT alone was less effective, and cholic acid alone was least effective in producing therapeutic liver repopulation. Overall, these findings should be helpful in guiding clinical strategies for liver-directed cell therapies in WD and other relevant disorders.
Previous studies established that RT by itself was insufficient for promoting transplanted cell proliferation in the healthy liver, although in combination with other perturbations, e.g., PH, IRP, hormones or carbon tetrachloride-induced hepatotoxicity, RT produced extensive liver repopulation in rats or mice over approximately three months (3,5,26). These manipulations share the common threads of hepatic oxidative stress and DNA damage with adverse manifestations including mechanisms of cell ploidy, cell cycling and cell death (27). In previous studies, PH was shown to produce oxidative stress in the liver, which resulted in decreased capacity for hepatocellular proliferation under growth factor stimulation conditions in vitro, as well as during liver repopulation conditions in vivo to explain how PH would synergize with RT and create favorable conditions for liver repopulation (20,22). On the other hand, in LEC rats with significant ongoing hepatic oxidative stress, RT by itself was beneficial for proliferation of transplanted cells and induction of additional oxidative stress through RT plus cholic acid exerted further synergistic benefits on proliferation of transplanted cells. This synergistic effect of RT and the hydrophobic bile salt, cholic acid, was in agreement with previous studies of bile salt-induced hepatotoxicity, e.g., short-term infusion of hydrophobic bile salt in rats, which produced acute perivenous liver injury (16), as well as addition to the cell culture medium of cultured hepatocytes, establishing induction of apoptotic cell death through subcellular mechanisms, such as endoplasmic reticulum stress-mediated or ceramide and protein kinase-dependent pathways (17–19). By contrast, hepatic preconditioning with RT-based strategies was less effective in LEC rats with copper toxicosis compared with healthy rats because fewer animals demonstrated successful correction of WD phenotype (10), whereas near-total liver repopulation was produced in the latter within 3-months following hepatic conditioning with either RT plus PH or RT plus IRP (3,5).
Accumulation of copper produces multiple hepatic changes, including oxidative stress and lipid peroxidation, as demonstrated here, and previously (28), with likely impact on mechanisms regulating hepatic preconditioning through further oxidative stress, DNA damage and other injuries. For instance, we observed different manifestations of RT and cholic acid-induced liver injury in healthy animals, where fatty change and necrosis were evident, while these changes were absent in LEC rats even though hepatic metabolism of lipids was abnormally regulated in these animals (29). However, over the long-term, exposure of LEC rat hepatocytes to RT and cholic acid did produce advantages for transplanted healthy cells. At present, we do not know whether administration of cholic acid over longer durations would have offered further advantages to transplanted hepatocytes. In previous studies, after transplantation of healthy hepatocytes in mice with impaired biliary phospholipid excretion due to P-glycoprotein deficiency, which causes accumulation of toxic bile salts and hepatocellular injury, continued administration of toxic bile salts over several months produced significant liver repopulation without clearance of transplanted cells (30), suggesting that longer use of cholic acid should be effective.
Other reasons for slower kinetics of liver repopulation in LEC rats should include direct effects of copper on transplanted cell proliferation, including through increased intracellular shunting of copper during its removal, as well as oxidation of extracellular matrix components, which was found to perturb survival of cultured hepatocytes with altered outside-in cell signaling involving NF-κB and other transcriptional factors (31).
The intracellular nature of signals that might regulate hepatic conditioning in the setting of chronic oxidative stress, such as in WD, are unknown at present, and are likely to be highly complex. For instance, we could speculate that hepatic preconditioning in the setting of copper toxicosis may benefit from the activation of inflammatory cytokines through Kupffer cell-mediated responses (32), although substantiation of this mechanism will require further studies. Similarly, onset of oxidative stress in the liver activated changes, such as increased hepatic expression of -glutamyltranspeptidase, which was associated with greater survival of cells, including resistance to further oxidative stress (33). How intracellular events may be altered during chronic oxidative stress is under active investigation with consideration of cell signaling mechanisms involving mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase 1/2, c-Jun N-terminal kinase (JNK) and p38 MAPK, regulation of NF-kB, or other processes capable of regulating cell cycling and/or cell death (34). Dissecting intracellular signaling pathways in the context of chronic copper toxicosis will likely be complex due to the possibility of redundancies and opposing subcellular responses in cellular pathways. Nonetheless, in short-term cell culture studies, copper has been demonstrated to increase expression of activating protein (AP)-1, cell cycle-regulated genes, e.g., c-Fos, c-Jun and c-Myc, as well as JNK and p38 MAPK, within the settings of oxidative stress (35,36). Also, oxidative preconditioning was found to protect against further metal toxicity, again through possible involvement of MAPK pathways (37), although development of these insights in the context of WD or other disorders will require studies in appropriate models, such as LEC rats. Such studies should help establish applications of differences in the responses to preconditioning regimens for cell therapy under disease-specific situations.
Funding: Supported in part by NIH grants R01 DK46952 and P30-DK41296
We thank Alan F. Hofmann for valuable discussions. This work was supported in part by NIH grants R01 DK46952 and P30 DK41296.