Spontaneous hepatic repopulation in transgenic mice expressing mutant human α1-antitrypsin by wild-type donor hepatocytes

Abstract

α1-Antitrypsin deficiency is an inherited condition that causes liver disease and emphysema. The normal function of this protein, which is synthesized by the liver, is to inhibit neutrophil elastase, a protease that degrades connective tissue of the lung. In the classical form of the disease, inefficient secretion of a mutant α1-antitrypsin protein (AAT-Z) results in its accumulation within hepatocytes and reduced protease inhibitor activity, resulting in liver injury and pulmonary emphysema. Because mutant protein accumulation increases hepatocyte cell stress, we investigated whether transplanted hepatocytes expressing wild-type AAT might have a competitive advantage relative to AAT-Z-expressing hepatocytes, using transgenic mice expressing human AAT-Z. Wild-type donor hepatocytes replaced 20%-98% of mutant host hepatocytes, and repopulation was accelerated by injection of an adenovector expressing hepatocyte growth factor. Spontaneous hepatic repopulation with engrafted hepatocytes occurred in the AAT-Z-expressing mice even in the absence of severe liver injury. Donor cells replaced both globule-containing and globule-devoid cells, indicating that both types of host hepatocytes display impaired proliferation relative to wild-type hepatocytes. These results suggest that wild-type hepatocyte transplantation may be therapeutic for AAT-Z liver disease and may provide an alternative to protein replacement for treating emphysema in AAT-ZZ individuals.

Figure 1. Kinetics of hepatic repopulation.

(A) Ad-HGF administration accelerated repopulation. ROSA26 mouse hepatocytes (1 × 10⁶) were transplanted into male PiZ mice without (upper row) or with (lower row) Ad-HGF (1 × 10¹¹ particles, i.v.). Liver sections were stained for E. coli β-gal (blue), and diastase plus PAS (magenta) to visualize AAT-Z globules. Scale bars: 100 μm. Data are from representative mice from each group (n = 6). (B) Repopulation was greater in male recipients. Male and female PiZ mice received Ad-HGF (n = 6). Hepatocyte transplantation and staining of liver sections were as in A. (C and D) Quantitative DNA PCR. Quantitative PCR for the E. coli lacZ gene was performed on DNA extracted from livers of recipient mice. Percentage of repopulation was calculated as described in the text. (C) Graphic presentation of data from experimental groups shown in A (mean ± SEM; n = 6 in each group), showing significantly higher repopulation in the Ad-HGF group at all time points (P < 0.05). (D) Data are from the experimental groups shown in B (mean ± SEM; n = 6 in each group), showing significantly higher repopulation in males 30, 60, and 90 days after transplantation (P < 0.05).

Figure 2. Depletion of hepatic human AAT-Z content and restoration of normal liver histology after repopulation.

(A) Male and female PiZ mice were transplanted with ROSA26 hepatocytes, with or without Ad-HGF administration (n = 6 in each group). Liver tissues were obtained by biopsy before and at various time points after transplantation and at the termination of the experiment. Hepatic human AAT-Z content was determined by Western blot/densitometry. Data (percentage of pretransplant values) are plotted against percentage of residual β-gal–negative host hepatocytes. The line of best fit is shown. (B) H&E staining of liver paraffin section after extensive (80%) hepatic repopulation by donor hepatocytes. (C–E) Masson’s trichrome–stained liver sections. (F–H) Sirius red–stained liver sections. (C and F) C57BL/6 mice; (D and G) untreated PiZ mice. (E and F) PiZ mice after 80% hepatic repopulation with wild-type hepatocytes. In Masson’s trichrome–stained sections, the area occupied by blue-stained collagen was quantified using the ImageJ program in conjunction with the Threshold Color plug-in. Scale bars: 100 μm.

Figure 3. Proliferation and apoptosis of hepatocytes after partial repopulation.

(A) Ki67 and β-gal immunofluorescent staining. Paraffin sections of formalin-fixed liver samples at various time points after transplantation (as in Figure 1) were subjected to immunofluorescent staining for Ki67 and E. coli β-gal. To visualize the nuclei, sections were mounted with Vectashield containing DAPI (Vector Laboratories). Representative liver sections from recipient PiZ mice, donor ROSA26 mice, and PiZ mouse livers partly repopulated with the ROSA26 hepatocytes are shown. (B–E) TUNEL staining. TUNEL staining was performed on cryosections of formalin-fixed livers from livers of PiZ mice (B), ROSA26 donor mice (C), and PiZ mouse livers after partial repopulation with the donor ROSA26 hepatocytes (E). D shows a section consecutive to that shown in E, stained for β-gal activity (blue) and AAT-Z globules (magenta). Scale bars: 100 μm.