PiZ mice have normal serum alanine aminotransferase (ALT), bilirubin, and albumin levels, but exhibit mild hepatic fibrosis.
Transgenic PiZ mice express human AAT-Z
from its native promoter elements (12
). Tissue distribution of transgene expression recapitulates that in humans (13
). However, because the endogenous mouse AAT is expressed normally, these mice do not have the loss-of-function phenotype of the human deficiency state. Serum ALT (25 ± 10 IU/l), bilirubin (0.25 ± 0.20 mg/dl), and albumin (3.8 ± 0.25 g/dl) levels in the PiZ mice were not significantly different from corresponding values in wild-type C57BL/6 mice (27 ± 11 IU/l, 0.21 ± 0.18 mg/dl, and 3.7 ± 0.28 g/dl, respectively). In this respect, these mice resembled the majority of human patients homozygous for the AAT-Z
mutation, in whom results of conventional serological liver function tests are within normal limits (1
H&E staining of liver sections showed normal liver architecture and occasional intralobular lymphocytic infiltration. However, Sirius red and Masson’s trichrome staining showed mild hepatic fibrosis, with fine strands extending into the lobules from perivascular regions, confirming previously published results (14
). As in human AAT-Z disease, diastase-resistant PAS-positive polymerized AAT-Z globules accumulate in the ER in clusters of hepatocytes, separated by globule-devoid cells. Consistent with previous reports (14
), male mice exhibited a larger number of globule-containing hepatocytes (data not shown).
AAT-Z globule–containing hepatocytes have AAT-Z transgene and mRNA content similar to that of globule-devoid cells, but AAT-Z protein load is greater.
To determine the transgene content of globule-containing and the globule-devoid cells, we performed LASER capture microscopy on diastase/PAS-stained liver sections. DNA PCR showed that the AAT-Z
transgene was present in both groups of hepatocytes. Quantitative RT-PCR revealed that both groups of hepatocytes expressed similar AAT-Z
mRNA levels (data not shown). In contrast, Western blot analysis showed a much higher AAT-Z content in the globule-containing hepatocytes, which was consistent with the results of Lindblad et al., who also showed that the globule-containing cells had a higher rate of apoptosis than globule-devoid hepatocytes (15
Together the results indicate that both globule-containing and globule-devoid cells express AAT-Z, but the globule-containing cells accumulate more polymerized AAT-Z, probably through posttranscriptional mechanisms. Despite the lesser accumulation of AAT-Z in globule-devoid hepatocytes, these cells may still be under sufficient intracellular stress, causing a proliferative disadvantage when in competition with transplanted wild-type hepatocytes (as shown below).
In vivo bioluminescence imaging showed spontaneous proliferation of transplanted wild-type donor hepatocytes in PiZ mouse livers.
To determine noninvasively whether wild-type donor hepatocytes proliferated in PiZ mouse livers, hepatocytes from C57BL/6 mice were transduced ex vivo using a recombinant lentivirus expressing firefly luciferase before transplantation. The transduction efficiency was approximately 30%. Fourteen days after transplantation, in vivo bioluminescence imaging showed that the transplanted cells were concentrated in the spleen. At later time points, marked increase in the signal over the liver indicated intrahepatic proliferation of engrafted hepatocytes (Supplemental Figure 1; supplemental material available online with this article; doi:
Wild-type hepatocytes engraft in PiZ mouse livers as single cells and proliferate spontaneously over time.
Three days after transplantation of β-gal–expressing ROSA26 mouse hepatocytes, the donor cells were engrafted as single hepatocytes, without site preference in relation to the AAT-Z globule–containing hepatocytes. After 30 days, proliferating clusters of the engrafted hepatocytes were found (Figure A). With progression of repopulation (Figure A), AAT-Z globule–containing hepatocytes declined, so that after 90 days, very few of these cells remained. In contrast, after transplantation of ROSA26-derived hepatocytes into congeneic C57BL/6 recipients, with or without the injection of an adenoviral vector expressing human hepatocyte growth factor (Ad-HGF), the engrafted cells remained as single cells or microclusters of 2–3 hepatocytes (data not shown).
Kinetics of hepatic repopulation.
Ad-HGF accelerated liver repopulation.
To test the hypothesis that mitotic stimulation should accelerate liver repopulation by enhancing the difference between the proliferative rates of AAT-Z–expressing host hepatocytes and the wild-type donor cells, we determined the time course of repopulation with or without Ad-HGF (1011 i.v.) administration in male PiZ mice. In one control group, Ad-HGF was replaced with an adenovector expressing an unrelated gene, UGT1A1 (Ad-UGT1A1). Morphometric analysis showed that hepatic repopulation by donor hepatocytes was significantly greater at all time points in the Ad-HGF group, but not in recipients receiving Ad-UGT1A1 (Figure A). Ninety days after transplantation, in Ad-HGF–treated recipients, 70%–98% of the hepatocytes were replaced by the donor cells, but the host bile duct epithelial cells and the nonparenchymal cells persisted (Figure A). The effects of Ad-HGF administration (Figure C) on repopulation were corroborated by quantitative DNA PCR of the LacZ gene in hepatocytes isolated from the donor and recipient livers. Repopulation was estimated assuming 2 × 105 cells being present per mg liver weight and 60% of these cells being hepatocytes.
Interestingly, even in groups receiving no adenovector or Ad-UGT1A1, 180 days after transplantation, liver repopulation was 70% ± 9.2% (mean ± SEM) and 59% ± 10%, respectively (Figure C).
Hepatic repopulation was more extensive in male recipient mice than in females.
Initial engraftment of hepatocytes was equally efficient in male and female recipients (3 days; Figure B). However, 30, 60, and 90 days after transplantation, hepatic repopulation in males was more extensive than in females (Figure B). The morphometric analysis was corroborated by quantitative DNA PCR of the LacZ gene in hepatocytes isolated from the donors and in the recipient liver.
Rudnick et al. (14
) reported higher levels of AAT-Z expression and a greater number of AAT-Z globule–containing hepatocytes in male PiZ mice than in females. Testosterone administration to female PiZ mice increased AAT-Z expression and the number of globule-containing hepatocytes (14
). Therefore, the accelerated repopulation in male PiZ mice may be related to the effect of endogenous androgens on AAT-Z expression. However, within each sex, we did not find any correlation between the percentage of globule-containing cells in the initial biopsy and the rate of liver repopulation.
Hepatic AAT-Z content was progressively depleted after liver repopulation.
We analyzed liver tissue samples from the male and female PiZ mice at various time points after transplantation of 1 × 106 hepatocytes from ROSA26 donors, with or without Ad-HGF (1 × 1011 particles) administration. Western blot with densitometric analysis of band intensities showed that PiZ content of the liver declined progressively following hepatocyte transplantation (Figure A).
Depletion of hepatic human AAT-Z content and restoration of normal liver histology after repopulation.
Liver histology and function after repopulation.
Following extensive repopulation, H&E-stained liver sections showed normal architecture and hepatocyte morphology (Figure B). In untreated PiZ mice, Masson’s trichrome (Figure , C–E) and Sirius red (Figure , F–H) staining showed mild hepatic fibrosis, which resolved after extensive liver repopulation. Hepatic collagen content was estimated by image analysis of the blue-stained collagen in liver sections. In wild-type C57BL/6 and PiZ mice. hepatic collagen content was 0.45% ± 0.28% and 3.23% ± 1.25%, respectively (mean ± SD; n = 6, P < 0.01). After 40%–60% and 70%–90% repopulation, the collagen content decreased to 0.94% ± 0.45% and 0.55% ± 0.35%, respectively (n = 6). In PiZ mice with extensive liver repopulation (>70%), hepatic collagen content was not significantly different from that of C57BL/6 controls (P > 0.2). Serum bilirubin, ALT, and albumin levels remained normal (data not shown).
After transplantation, the donor hepatocytes divided more frequently, whereas the host hepatocytes exhibited increased apoptosis.
Progressive repopulation of the recipient mouse livers, without a change in the total liver size, suggested that the donor cell proliferation was counterbalanced by attrition of host hepatocytes, probably by apoptosis. To verify this, we quantified the number of dividing donor and host cells by immunofluorescent staining of the proliferation marker Ki67 and the number of host and donor cells undergoing apoptosis by TUNEL staining. For each section, 6 nonoverlapping fields, containing approximately 6000 hepatocytes, were counted. Dual immunofluorescent staining showed that resting PiZ mouse livers and livers from ROSA26 donor mice contained 3.8 ± 0.7 and 3.0 ± 0.8 Ki67-positive cells per 1000 (Figure A; n
= 6, mean ± SD). After 40%–60% repopulation of the liver, the number of Ki67-positive cells increased (10 ± 2.2 per 1000) in the β-gal–positive donor cell clusters (Figure A), but not in the host hepatocytes (2.5 ± 0.8 per 1000) (n
= 6, P
< 0.02). TUNEL staining showed a higher level of apoptotic cells (4 ± 1.8 per 1000) in untreated PiZ mouse livers (Figure B) than in wild-type C57BL/6 mice (1.8 ± 0.5 per 1000) (Figure C). There was no increase in the number of TUNEL-positive cells in nontransplanted male and female PiZ mice receiving tacrolimus (1 mg/kg daily). After partial liver repopulation, the frequency of TUNEL-positive host hepatocytes increased 3.5-fold (14 ± 3.2 per 1000, n
= 6, P
< 0.01) over that in the untreated PiZ mice. No increase in TUNEL staining was found in the β-gal–positive donor cell clusters (1.6 ± 0.8 per 1000; Figure , D and E). These results suggest that the engrafted cells proliferated at a greater rate in the host PiZ mouse livers than in livers of wild-type mice, probably via growth signals received from the host. The increased apoptosis of host hepatocytes combined with greater proliferation of donor cells resulted in progressive repopulation of the host liver, while the liver size remained unaltered, consistent with tight physiological control of the liver to body size ratio. This phenomenon is reminiscent of the cell-cell competition observed during Drosophila wing development, where the proximity of more mitotically active cells causes death of less mitotically competent, but otherwise viable cells (16
). This process is fundamentally different from liver repopulation in urinary plasminogen activator (uPA) transgenic mice and FAH-deficient mice, where the death of host hepatocytes results from intrinsic abnormalities and does not require the presence of wild-type donor cells (8
). Notably, after extensive repopulation, serological tests for hepatocyte function and liver histology remained normal.
Proliferation and apoptosis of hepatocytes after partial repopulation.
In summary, we show that engrafted wild-type hepatocytes proliferate preferentially over the recipient PiZ mouse hepatocytes. This is associated with enhanced apoptosis of the host hepatocytes, hepatic repopulation with donor hepatocytes, and resolution of the liver fibrosis that occurs in untreated PiZ mice. The repopulation was more extensive in male recipients and was accelerated by mitotic stimulation of hepatocytes. Our findings suggest that liver repopulation with transplanted normal allogeneic hepatocytes could be effective therapy for a subgroup of AAT-ZZ patients with liver disease and as an alternative to protein replacement for emphysema, even in the absence of severe liver disease.