1 Marion Bessin Liver Research Center, 2 Comprehensive Cancer Research Center, and Departments of 3 Medicine, 4 Cell Biology, 5 Pathology, and 6 Radiology, Albert Einstein College of Medicine, Bronx, New York 10461
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ABSTRACT |
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Cell transplantation
into hepatic sinusoids, which is necessary for liver repopulation,
could cause hepatic ischemia. To examine the effects of cell
transplantation on host hepatocytes, we transplanted Fisher 344 rat
hepatocytes into syngeneic dipeptidyl peptidase IV-deficient rats.
Within 24 h of cell transplantation, areas of ischemic necrosis,
along with transient disruption of gap junctions, appeared in the
liver. Moreover, host hepatocytes expressed -glutamyl transpeptidase
(GGT) extensively, which was observed even 2 years after cell
transplantation. GGT expression was not associated with
-fetoprotein
activation, which is present in progenitor cells. Increased GGT
expression was apparent after transplantation of nonparenchymal cells
and latex beads but not after injection of saline, fragmented
hepatocytes, hepatocyte growth factor, or turpentine. Some host
hepatocytes exhibited apoptosis, as well as DNA synthesis, between 24 and 48 h after cell transplantation. Changes in gap junctions, GGT
expression, DNA synthesis, and apoptosis after cell transplantation
were prevented by vasodilators. The findings indicated the onset of
ischemic liver injury after cell transplantation. These hepatic
perturbations must be considered when transplanted cells are utilized
as reporters for biological studies.
hepatocyte; ischemia; injury; gene expression; vasodilatation
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INTRODUCTION |
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TRANSPLANTED HEPATOCYTES integrate in the liver (20), repopulate the liver extensively in appropriate situations (24, 31, 37), and retain excellent function (22). In view of the therapeutic potential of hepatocyte transplantation (17), it is necessary to establish detailed mechanisms concerning liver repopulation with transplanted cells. Recent studies (21, 34) established that only a fraction of transplanted hepatocytes survive in the host liver. Although hepatocytes deposited in hepatic sinusoids integrated in the liver parenchyma, cells in portal spaces were cleared (21). In addition, it became apparent that there is temporary occlusion of portal vessels with cell emboli, which has the potential to cause hepatic ischemia. Subsequently, several days were required for transplanted cells to join host hepatocytes in the liver plate with the formation of conjoint plasma membrane structures. These findings indicate that the host liver undergoes perturbations after cell transplantation in hepatic sinusoids.
During our ongoing analysis of liver repopulation, we observed
unexpected -glutamyl transpeptidase (GGT) expression in the host
liver. In the normal adult liver, GGT is only expressed in biliary
cells, whereas GGT is expressed in fetal hepatoblasts, precancerous or
cancerous liver nodules, and malignant hepatocyte-derived cell lines,
as well as "oval cells" arising in response to liver injury or
carcinogenic treatments (2, 14, 23, 32). We were analyzing
the fate of genetically marked cells in animals treated with the
hepatotoxin D-galactosamine (GalN), which causes activation
of progenitor liver cells expressing GGT (7). The hypothesis was that transplanted progenitor cells will differentiate into mature hepatocytes in a permissive microenvironment, and this was
demonstrated with both pancreatic and hepatic epithelial cells after
transplantation into the liver (8). Other studies examined
the fate of transplanted hepatocytes in GalN-induced acute liver
failure (19). Our expectation was that GGT expression might be activated in transplanted progenitor cells, but these cells
matured into hepatocytes. However, despite maturation of progenitor
cells into hepatocytes, we found that GGT expression was induced
extensively in the host liver after cell transplantation. This prompted
us to undertake further analysis of mechanisms underlying this
observation. We used dipeptidyl peptidase IV deficient (DPPIV
) Fischer 344 (F344) rats as hepatocyte recipients, which facilitates the
localization of syngeneic normal hepatocytes in the liver, as
previously described (35). Because one mechanism
underlying GGT expression in host hepatocytes could involve induction
of hepatic ischemia, studies were conducted to analyze additional changes relevant to this process after cell transplantation.
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MATERIALS AND METHODS |
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Cells.
Primary hepatocytes were isolated by in situ collagenase perfusion of
the liver as described previously (35). Cell viability was
documented by trypan blue dye exclusion and attachment to tissue
culture plastic in RPMI 1640 medium containing penicillin, streptomycin, amphotericin B, and 10% fetal bovine serum (FBS) (GIBCO,
Grand Island, NY). FBS was from Hyclone Laboratories (Logan, UT).
Collagenase was from Boehringer Mannheim (Indianapolis, IN). A cell
line derived from nonparenchymal cells of the young rat liver,
designated G7FNRL cells, that contains a bacterial -galactosidase has been described previously (30). These cells were
cultured in RPMI 1640 medium with penicillin, streptomycin,
amphotericin B, and 10% FBS.
Animals.
Male F344 rats were obtained from the National Cancer Institute. The
Special Animals Core of the Marion Bessin Liver Research Center
provided DPPIV F344 rats weighing 120-150 g. Both male and
female rats were used for the studies. Approximately 90 rats were
utilized for the studies. The animals were housed under 14:10-h light/dark cycles with standard pelleted rodent diet and water ad
libitum. The Animal Care and Use Committee at Albert Einstein College
of Medicine approved the experimental protocols, and animal use was in
accordance with the Guide for the Care and Use of Laboratory Animals [DHEW Publication No. (NIH) 85-23, revised 1985, Office of Science and Health Reports, DRR/NIH, Bethesda, MD 20505].
Histochemical assays.
Tissues were frozen in methylbutane cooled to 70°C, and
5-µm-thick cryostat sections were prepared. DPPIV activity was
visualized after fixing sections in cold chloroform and acetone (1:1
vol/vol) for 10 min followed by incubation with
glycyl-L-proline-4-methoxy-2-naphthylamide substrate in the
presence of 1 mg/ml fast blue BB salt for 30 min, as described
previously (26). ATPase activity was used to colocalize
bile canaliculi in the host and transplanted hepatocytes as reported
previously (18). GGT activity was detected in tissue sections or cells fixed with ethanol-glacial acetic acid for 10 min at
20°C, according to Rutenberg et al. (38). The overall distribution of GGT activity in tissues was classified as being either
grade 0 (absent), grade 1+ (scanty; an
occasional cell per high power field positive), grade 2+
(moderate; 20-50 cells per high power field positive), or
grade 3+ (extensive; >50 cells per high power field
positive). Hepatic gap junctions were localized by immunostaining with
the 7C6,
Cx32 antibody, as described previously (20).
In some studies, tissues were stained histochemically for DPPIV
activity, followed by Cx32 immunostaining. Tissues were counterstained
with hematoxylin or methyl green as appropriate.
Cellular glutathione and catalase content.
All chemicals were from Sigma Chemical. Cells were harvested and stored
at 80°C in 5% salicylic acid. For assays, cells were thawed to
4°C and disrupted by ultrasonication. Cell debris were eliminated by
pelleting at 10,000 g for 10 min at 4°C. The supernatant was assayed for total glutathione content (1). To 100 µl
supernatant, 800 µl of 0.3 mM nicotinamide adenine dinucleotide
phosphate, reduced form, 100 µl of 6 mM DTNB, and 0.5 U glutathione
reductase were added. Changes in absorbance over 2 min at 412 nm were
read spectrophotometrically. Glutathione standards were prepared with 100 µM stock solution in linear range. Catalase activity was
determined by a method described previously (27). Briefly,
frozen cells were thawed on ice, ultrasonicated, and then centrifuged
at 10,000 g for 10 min at 4°C. Catalase activity was
measured in the supernatant by adding 3 ml of
H2O2-phosphate buffer and 10-40 µl
sample in a silica cuvette, and time (t) for change in
optical density from 0.450 to 0.400 was determined at 240 nm at room
temperature. The catalase activity was calculated by the formula
17/t = units/assay mixture. Protein content was assayed
in aliquots using the Bradford assay. Each condition was in triplicate.
Quantitation of GGT activity in tissues. A commercial assay kit was used (Diagnostics Procedure 545, Sigma Chemical). Preweighed liver tissues were homogenized in PBS, pH 7.4, and incubated with L-glutamyl nitroanilide substrate for 37°C for 20 min. The reactions were conducted according to the manufacturer's suggested protocol and stopped by the addition of glacial acetic acid. Sodium nitrite, ammonium sulfamate, and naphthylethylenediamine were added, and resultant absorbance was measured at 540 nm for each condition against blanks containing equal amounts of unreacted liver tissue. The reaction in blank tubes was stopped by adding glacial acetic acid to the substrate before the addition of liver homogenate and other reagents. A standard curve was plotted using GTP calibration solution (Sigma Chemical, no. 545-10).
DNA synthesis assays. Animals were given 0.5 mCi/kg body wt [3H]thymidine (specific activity, 70 Ci/mmol; ICN, Irvine, CA) and 50 mg/kg ip bromodeoxyuridine (BrdU; Boehringer Mannheim, Indianapolis, IN) 1 and 2 h before death, respectively. To demonstrate mitotic spindles, animals were given colchicine (Sigma Chemical) in a dose of 0.5 mg/kg body wt 2 h before death. To detect [3H]thymidine incorporation, tissue sections were autoradiographed for 4 wk with NTB-2 emulsion (Eastman Kodak, Rochester, NY), as described previously (18). To detect BrdU incorporation, cryostat sections were fixed in cold ethanol or ethanol-acetic acid (99:1 vol/vol) and blocked with 2% rabbit serum followed by incubation for 1 h with anti-BrdU (Amersham Life Sciences, North Chicago, IL). Antibody binding was detected by a supersensitive multilink antibody system, using the peroxidase reporter (BioGenex Laboratories, San Ramon, CA), followed by color development with Vectastain (Vector Laboratories, Burlingame, CA). To localize BrdU incorporation in GGT-positive cells, tissues were first stained for GGT activity and then subjected to BrdU immunostaining. DNA synthesis in transplanted cells was analyzed by DPPIV staining followed by BrdU immunostaining.
In situ hybridization for -fetoprotein mRNA.
The recombinant plasmid pBAF700 containing
-fetoprotein (AFP) cDNA
sequences derived from fetal rat mRNAs was originally provided by Dr.
N. Fausto (7). After linearization with restriction enzymes, 35S-labeled anti-sense and sense riboprobes were
obtained with appropriate RNA polymerases. In situ hybridization was
performed on 5-µm-thick paraformaldehyde-fixed cryostat sections as
described (7). Hybridized sections were autoradiographed
at 4°C with NTB-2 emulsion (Eastman Kodak). Tissue sections from
frozen fetal rat liver were included as positive controls.
In situ demonstration of apoptosis. Cryostat tissue sections of 5-µm thickness were analyzed using a commercial kit with peroxidase detection (Boehringer Mannheim). The enzymatic reaction utilizes terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay (12). Tissues were fixed in cold ethanol for 10 min, followed by processing according to the manufacturer's suggested protocol. The assay identifies DNA strand breaks that occur during apoptosis by labeling free 3'-OH termini with modified nucleotides. Tissues from at least two animals were analyzed for each time point reported.
Statistical methods.
Data are presented as means ± SE or SD. The significance of
differences was analyzed with the Student's t-test,
Mann-Whitney rank-sum test, or 2 test using SigmaStat
2.0 software (Jandel Scientific, San Rafael, CA). P < 0.05 was considered significant.
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RESULTS |
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Host hepatocytes expressed GGT immediately after cell
transplantation.
When injected into the spleen, hepatocytes migrated immediately
to the liver and were found in hepatic sinusoids within 1 h, as
documented previously (21, 35). We found that in contrast with the normal adult liver, cell transplantation caused extensive GGT
expression in host hepatocytes. This became apparent as early as 2 h after cell transplantation (Fig. 1).
Hepatocytes expressing GGT were observed primarily in periportal areas,
although GGT-positive cells were also present in midzonal positions of
the liver lobule. There was a characteristic distribution of GGT
expression, with hepatocytes becoming positive in areas of the liver
distal to transplanted cells. On the other hand, transplanted cells
themselves remained GGT negative. Surprisingly, extensive GGT
expression in host hepatocytes was found in all tissues from hepatocyte
recipients, including at early (2-24 h and 2-4 days) and
intermediate times (7 days and 3 wk), as well as late times (3, 11, and
24 mo). Indeed, tissues analyzed at 11 mo after cell transplantation
still showed significant GGT expression in hepatocytes, similar to that
observed at earlier times (Fig. 1D). Moreover, the overall
pattern of GGT expression was essentially unchanged with time.
GGT-positive cells remained distributed in periportal areas of the
liver with no GGT activity in perivenous areas. In addition, the
proportion of cells containing GGT activity was similar at early and
late times, with GGT expression ranging from grade 2+ to
3+ on histological analysis. Together, these findings
indicated that once GGT expression was induced in the liver,
hepatocytes expressed this activity permanently.
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Early GGT expression is associated with evidence of ischemic
events, including disruption of hepatic gap junctions.
Portal venography immediately after cell transplantation showed
attenuation of major portal vein radicles (Fig.
3). These findings were similar to those
reported elsewhere (21) with restoration of normal portal
vasculature within 1 day after cell transplantation.
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DNA synthesis in response to cell transplantation.
Analysis of hepatic DNA synthesis with either
[3H]thymidine or BrdU incorporation showed similar
kinetics in host hepatocytes. Rats injected with saline alone showed no
evidence for significant DNA synthesis, similar to untreated control
animals (0.1-0.2% labeled hepatocytes). In contrast, host
hepatocytes showed unscheduled DNA synthesis, as indicated by BrdU
labeling (Fig. 5). Scattered host
hepatocytes with or without GGT expression and other adjacent GGT-positive hepatocytes also showed DNA synthesis. To determine the
kinetics of this change, a series of animals were analyzed. Hepatic DNA
synthesis first became apparent at 24 h (2 ± 2% labeling), became most pronounced at 48 h (5 ± 3% labeling,
P < 0.001, 2 test), and was not
detected subsequent to 72 h after cell transplantation. Interestingly, transplanted hepatocytes were not observed to be undergoing DNA synthesis at these early times, i.e., 24 or 48 h
after cell transplantation (Fig. 5A). The findings were
verified by analysis of BrdU labeling in tissue sections stained first for DPPIV activity. Moreover, although DNA synthesis was not analyzed in the long term, the number of transplanted cells did not change in
recipients between 3 and 11 mo.
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Absence of AFP expression in liver after cell transplantation.
Expression of GGT and DNA synthesis in periportal cells led us to
consider whether progenitor cells had become activated. Therefore, we
analyzed AFP mRNA expression in the liver by in situ hybridization.
Although the control fetal rat liver contained cells with abundant AFP
mRNA expression, the liver of adult rats receiving transplanted
hepatocytes never showed hybridization signals at 12, 24, 48, or 3 wk
of cell transplantation (Fig. 6). The
experiments were repeated twice with similar data. Moreover, we did not
observe oval cells or expansions of biliary cells in the normal liver
after cell transplantation. These findings are consistent with an
absence of progenitor cell activation.
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Further evidence for injury to host hepatocytes.
Our observations concerning the presence of mitoses in
colchicine-pretreated animals were interesting. Despite DNA synthesis in the liver after hepatocyte transplantation, cells in mitosis were
not observed in metaphase. On the other hand, we found scattered GGT-positive and GGT-negative host hepatocytes with nuclear
fragmentation (Fig. 7). Colocalization
studies showed that DPPIV-positive transplanted cells neither
incorporated BrdU nor exhibited nuclear fragmentation at these times.
Notably, such nuclear fragmentation was not observed in animals treated
with nitroglycerine before cell transplantation. To determine whether
the change in host cells was compatible with the onset of apoptosis, in
situ TUNEL assays were performed. These studies showed increased
apoptosis rates in the liver 24 and 48 h after cell
transplantation. In the normal control liver, only an occasional cell
(1-2 cells/section) showed apoptosis. In contrast, 5 ± 3 apoptotic cells were observed per high power field (×100) in
hepatocyte recipients (P < 0.01, t-test).
Furthermore, hepatocyte apoptosis was not observed in animals
pretreated with nitroglycerine.
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Biological effects of GGT expression in hepatocytes. To determine the significance of GGT expression, hepatocytes were isolated from F344 rats that 3 wk previously received 5 × 107 cells via the spleen. Trypan blue dye exclusion showed that cell viability was >85%, similar to cells from control animals not treated with cell transplantation. Analysis of GGT expression in cytospun preparations showed that 30-40% of hepatocytes from transplanted animals were GGT positive. In contrast, hepatocytes from control animals did not express GGT. The glutathione content was greater in hepatocytes from transplanted animals (39 ± 1 µM glutathione/µg protein) compared with hepatocytes from control animals (21 ± 1 µM glutathione/µg protein, P < 0.05), whereas catalase activity was not different (87 ± 8 vs. 98 ± 7 units/mg protein). Analysis of DNA synthesis in cultured hepatocytes showed that cells from transplanted animals responded to hHGF with four- to ninefold increases in DNA synthesis. In contrast, hepatocytes from control animals showed 6- to 15-fold increases in DNA synthesis after stimulation with hHGF, which was not significantly different from transplanted animals.
On the other hand, hepatocytes from transplanted animals were more resistant to oxidant injury with t-BuOOH (Table 3). The data shown are from triplicate conditions and were obtained by exposing cells to t-BuOOH for 1 h after cells had been in culture for 48 h. Hepatocytes from animals treated with cell transplantation showed greater viability without any exposure to t-BuOOH and showed significantly greater resistance to 50 and 100 µM concentrations of t-BuOOH, which has been well characterized for its toxic effects on hepatocytes (29). Together, these findings indicate that activation of GGT expression in the liver was associated with greater resistance of cells to oxidative injury.
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DISCUSSION |
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We consider it most likely that mechanisms related to hypoxia or ischemia-reperfusion were responsible for GGT activation, loss of gap junction activity, and other changes after cell transplantation. Embolization of cells in hepatic sinusoids would be consistent with hypoperfusion, as well as hypoxia in areas distal to transplanted cells. Subsequent restoration of the microcirculation with resumption of blood flow would trigger ischemia-reperfusion events.
Acute and chronic hypoxia is known to induce hepatic GGT expression (40). Hepatic ischemia leads to peripheral release of GGT with peak blood levels between 20 and 30 h after restoration of hepatic blood flow (25), although our studies showed that hepatocyte membrane-bound GGT is expressed much earlier (within 2 h). GGT was activated in the liver by transplantation of relatively few cells, such as 1,000,000- 5,000,000 cells. We believe this to indicate that extensive occlusions of portal vein radicles are not required for inducing ischemic changes in the liver. The ability to reproduce induction of GGT by inert latex beads and its abrogation by vasodilation with nitroglycerine and phentolamine, which presumably restored blood flow in distal microcirculations, are also in agreement with ischemia-reperfusion-type mechanisms.
GGT is localized in cell membrane domains in many extrahepatic tissues, including, for instance, kidney, pancreas, spleen, heart, and brain, and is thought to play a role in amino acid transport across cell membranes (14). Although drugs, such as phenobarbitone and phenytoin, as well as alcohol, are known to induce GGT expression, the precise pathophysiological significance of this change is unclear, especially because of complex alterations accompanying these perturbations (39, 45). GGT expression may be regulated by multiple factors, including animal strain, hormones, cell differentiation states, and tissue-specific mechanisms, as suggested by studies (4, 5, 9, 10, 42, 48) in intact animals and cultured cells. The peculiar susceptibility of female F344 rats to GGT expression has previously been reported (4), although in our experience, GGT expression was observed after cell transplantation in both male and female rats, male mice, and male rabbits, exhibiting the broad nature of the finding.
Use of AFP expression as a marker for progenitor cell activation suggested that this did not account for GGT expression in our hepatocyte recipients. Also, there was no morphological evidence for progenitor cell activation in the liver, such as the appearance of oval cells. Recent studies (5, 9) demonstrated that GGT expression may be regulated by cell differentiation states at the transcriptional level, through promoter interactions with specific cellular factors. These studies indicate that GGT expression is increased when cells undergo differentiation rather than dedifferentiation, which is again compatible with the absence of progenitor cell activation in our animals. Among other regulatory mechanisms, it has been shown that glucocorticoids induce and thyroid hormones suppress GGT expression in the liver by transcriptional or other mechanisms, including alterations in cell differentiation states (9). We do not know whether cell transplantation altered the local availability, incorporation, or metabolism of these hormones in the liver.
GGT expression can also be induced by growth factor activation (10, 42). We did not study changes in the local release of specific growth factors or cytokines after cell transplantation, although studies by Yazigi et al. (47) have suggested that HGF is expressed in areas adjacent to transplanted hepatocytes. Nonetheless, we found that hHGF infusion alone did not induce GGT expression after cell transplantation. Similarly, our studies using turpentine to induce an acute phase response along with cytokine release (3, 15), including that of interleukin-6, which plays a role in liver regeneration (6), showed no activation of GGT. Our inability to induce GGT expression by injection of disrupted cells also suggested that soluble factors released from hepatocytes themselves were unlikely to be responsible for this change.
Previous analysis (46) of GGT-positive hepatocytes isolated from hepatic nodules arising in response to carcinogenic treatments demonstrated that these cells possessed greater proliferative capacity. However, hepatocytes influenced to express GGT in our studies did not proliferate in the liver. Indeed, our findings are most compatible with the persistence of GGT-positive hepatocytes without change in the overall mass of these cells, similar to the fate of transplanted hepatocytes in animals (16). Although we observed unscheduled DNA synthesis in host hepatocytes after cell transplantation, DNA synthesis was observed in GGT-negative hepatocytes, as well as GGT-positive hepatocytes. Moreover, we have found no evidence for oncogenesis in several F344 rats subjected to cell transplantation during up to two years of observation (n = 12; S. Gupta, unpublished observations).
The prolonged duration of GGT expression in the liver after cell transplantation was most intriguing. We found that increased hepatic GGT expression was still present at 2 years after a single session of cell transplantation. Although we do not know the basis for indefinite persistence of GGT expression in hepatocytes, previous studies (41) showed that hepatocytes expressing GGT were protected against glutathione depletion and oxidative stress. Thus one could speculate that if ischemia-reperfusion-related oxidative stress occurred in the liver after cell transplantation, GGT expression might represent a protective event, which could increase cell survival. Our findings were in agreement with this possibility. In hepatocytes isolated from animals subjected to cell transplantation, greater resistance to t-BuOOH injury is compatible with survival advantages, rather than increased proliferation of cells, as indicated by the absence of greater DNA synthesis in these hepatocytes compared with hepatocytes from the unperturbed normal rat liver.
We found it remarkable that the activity of Cx32, which is a major constituent of hepatic gap junctions, was rapidly, albeit transiently, altered after hepatocyte transplantation. Although hepatic Cx32 expression is regulated in the context of partial hepatectomy, drug-induced neoplasia, and other situations (33, 43, 44, 49), changes in Cx32 expression after ischemia-reperfusion are directly relevant to our observations. Recent studies (13), published subsequent to the performance of our experiments, showed that after ischemia hepatic Cx32 expression is attenuated within 1- 4 h after reperfusion. Similarly, cell transplantation in the presence of vasodilators prevented the loss of hepatic Cx32 expression in the liver. Our findings concerning Cx32 expression are in agreement with the onset of ischemia-reperfusion injury in the liver after hepatocyte transplantation. Angiographic analysis indicated attenuation of portal radicles after cell transplantation that is transient (21).
Findings in this study will be helpful in understanding changes occurring during liver repopulation and have possible implications in basic hepatic biology and pathophysiology. For instance, it should be of interest to analyze how cells induced to express GGT after hepatocyte transplantation differ from GGT-positive cells arising in response to carcinogenic treatments. Fractionation of GGT-positive hepatocytes from the liver not subjected to carcinogenic treatments should help advance our knowledge in this area. Our findings should also be helpful in defining whether changes in the host liver after cell transplantation could be of significance in cell therapy, such as in acute liver failure. Could it be that in the setting of significant liver injury, e.g., after drug or other toxicity, cell transplantation could worsen the situation initially by inducing further ischemia-reperfusion injury? Finally, our findings stress the need for caution in conducting and interpreting cell transplantation studies utilizing GGT as a marker, especially where analysis of the fate of progenitor liver cells is concerned (28).
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ACKNOWLEDGEMENTS |
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We thank Dr. M. Rojkind for advice concerning use of turpentine in animals, Dr. E. Hertzberg for providing the Cx32 antibody, and Dr. Ralph Schwall (Genentech) for providing hHGF.
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FOOTNOTES |
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The work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grants RO1 DK-46952, RO1 DK-17609, and P30 DK-41296, and an award from the Irma T. Hirschl Trust.
Address for reprint requests and other correspondence: S. Gupta, Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Ullmann 625, 1300 Morris Park Ave., Bronx, NY 10461 (E-mail: sanjvgupta{at}pol.net).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 29 June 1999; accepted in final form 13 April 2000.
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