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Vitamin A storage in hepatic stellate cells in the regenerating rat liverWith special reference to zonal heterogeneity.

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THE ANATOMICAL RECORD PART A 286A:899 –907 (2005)
Vitamin A Storage in Hepatic Stellate
Cells in the Regenerating Rat Liver:
With Special Reference to Zonal
Heterogeneity
NOBUYO HIGASHI,1 MITSURU SATO,1 NAOSUKE KOJIMA,1
TOSHIAKI IRIE,1 KOICHI KAWAMURA,2 AYAKO MABUCHI,3 AND
HARUKI SENOO1*
1
Department of Cell Biology and Histology, Akita University School of Medicine,
Akita, Japan
2
Department of Cellular and Organ Pathology, Akita University School of Medicine,
Akita, Japan
3
Department of Physiology, University of Otago, Dunedin, New Zealand
ABSTRACT
Under physiological conditions, hepatic stellate cells (HSCs) within liver lobules store about 80% of the total body
vitamin A in lipid droplets in their cytoplasm, and these cells show zonal heterogeneity in terms of vitamin A-storing
capacity. Vitamin A is essential for the growth and differentiation of cells, and it is well known that liver cells including
HSCs show a remarkable growth capacity after partial hepatectomy (PHx). However, the status of vitamin A storage
in HSCs in the liver regeneration is not yet known. Therefore, we conducted the present study to examine vitamin A
storage in these cells during liver regeneration. Morphometry at the electron microscopic level, fluorescence microscopy
for vitamin A autofluorescence, and immunofluorescence microscopy for desmin and ␣-smooth muscle actin (␣-SMA)
were performed on sections of liver from male Wistar strain rats at various times after the animal had been subjected
to 70% PHx. The mean area of vitamin A-storing lipid droplets per HSC gradually decreased toward 3 days after PHx,
and then returned to normal within 14 days after it. However, the heterogeneity of vitamin A-storing lipid droplet area
per HSC within the hepatic lobule disappeared after PHx and did not return to normal by 14 days thereafter, even
though the liver volume had returned to normal. These results suggest that HSCs alter their vitamin A-storing capacity
during liver regeneration and that the recovery of vitamin A homeostasis requires a much longer time than that for liver
volume. © 2005 Wiley-Liss, Inc.
Key words: hepatic stellate cell; vitamin A; liver regeneration; partial hepatectomy; morphometry
Hepatic stellate cells (HSCs; also called vitamin A-storing cells, fat-storing cells, lipocytes, and interstitial cells)
are located in the perisinusoidal space of Disse and extend
their thin fibrillar processes into this space (Wake, 1971,
1980; Imai and Senoo, 1998, 2000; Imai et al., 2000a).
Under physiological conditions, the HSCs store about 80%
of the total vitamin A in the whole body as retinyl esters in
lipid droplets in their cytoplasm, and they show heterogeneity in their vitamin A-storing capacity within the liver
lobules (Wake and Sato, 1993; Zou et al., 1998). These cells
also play a pivotal role in the regulation of vitamin A
homeostasis (Senoo and Wake, 1985; Blomhoff et al., 1990;
Senoo et al., 1990, 1993a, 1993b; Blomhoff and Wake,
1991; Imai et al., 2000b). Earlier we reported the existence
of a gradient of vitamin A-storing capacity in the liver and
found that it was not dependent on the vitamin A amount
in the organ (Higashi and Senoo, 2003). This gradient was
expressed as a symmetrical biphasic distribution starting
at the periportal zone, peaking at the middle zone, and
©
2005 WILEY-LISS, INC.
sloping down toward the central zone in the liver lobule.
Vitamin A is essential for the growth and differentiation
of cells (Blomhoff, 1994; Sporn et al., 1994; Chambon,
1996), and it is well known that liver cells including HSCs
show a remarkable growth capacity after partial hepatec-
Grant sponsor: Grants-in-Aid for Young Scientists, Ministry of
Education, Culture, Sports, Science, and Technology of Japan;
Grant number: B 16790118.
*Correspondence to: Haruki Senoo, Department of Cell Biology
and Histology, Akita University School of Medicine, 1-1-1 Hondo,
Akita 010-8543, Japan. Fax: 81-18-834-7808.
E-mail: senoo@ipc.akita-u.ac.jp
Received 27 February 2005; Accepted 4 June 2005
DOI 10.1002/ar.a.20230
Published online 7 August 2005 in Wiley InterScience
(www.interscience.wiley.com).
900
HIGASHI ET AL.
tomy (PHx) (Michalopoulos and DeFrances, 1997; Tub,
2004). Moreover, it has also been reported that the recovery from liver damage including 70% PHx is influenced by
the content of vitamin A in the liver (Hauswirth, 1987; Hu
et al., 1994; Evarts et al., 1995; Ozeki and Tsukamoto,
1999). Storing vitamin A is one of the most important
functions of HSCs. The influence of liver regeneration,
especially regeneration after partial hepatectomy, on
other functions of HSCs has been examined from various
aspects. For example, the start point of DNA synthesis of
HSCs (Tanaka et al., 1990); alterations in the amounts of
protein products, including cytokines, enzymes, and transcription factors (Marsden et al., 1992; Watanabe et al.,
1998; Ujiki et al., 2000; Asahina et al., 2002); and cell-cell
interactions between HSCs and hepatocytes (Mabuchi et
al., 2004) have been already assessed in the regenerating
liver after 70% PHx. The fact that hepatocyte-HSC interaction in the early stages after PHx (1-3 days) is intimately involved in the regeneration process (Mabuchi et
al., 2004) has particularly shown the importance of the
role of HSCs during liver regeneration (Balabaud et al.,
2004).
However, the status of vitamin A storage in HSCs during liver regeneration is not yet known. To address this
question, we conducted the present study by focusing on
the heterogeneity of vitamin A-storing capacity within the
liver lobules.
MATERIALS AND METHODS
Animals
Male Wistar strain rats weighing 150 –160 g were obtained from a commercial source (Clea Japan, Tokyo, Japan). The rats had been maintained on a standard cake
diet (Clea Japan). The protocols used in this study for
specimen preparation were previously approved by the
Animal Research Committee, Akita University School of
Medicine. All subsequent specimen preparation adhered
to the university’s guidelines for animal experimentation.
Experimental Groups
For each examination in this study, six experimental
groups (n ⫽ 3 for each) received 70% PHx were used: 1 day
(PHx1), 3 days (PHx3), 5 days (PHx5), 7 days (PHx7), and
14 days (PHx14) after 70% partial hepatectomy. Controls
were prepared (3 days after the sham-operation; for the
sham-operation, we opened the abdomen and the liver was
pull out of the abdominal cavity and was put into the
original position without partial hepatectomy).
Surgical Techniques
Seventy percent partial hepatectomy was performed by
using a modification of the technique described by Higgins
and Anderson (1931). Control rats underwent a shamoperation as mentioned above.
Electron Microscopy
For examination by transmission electron microscopy
(TEM), the livers were perfused with 1.5% glutaraldehyde
in 0.062% M cacodylate buffer, pH 7.4, containing 1%
sucrose for 1 or 2 min through the portal vein. After
perfusion, tissue blocks (2 mm ⫻ 2 mm ⫻ 2 mm) were
prepared as described previously (Senoo et al., 1999). For
morphological methods, namely, electron microscopy, flu-
orescence microscopy, and immunofluorescence microscopy, we took specimens by systematic random sampling.
This sampling method minimized the measurement influences.
Morphometry
To create zonal maps of the liver lobule, we examined by
light microscopy semithin sections of specimens embedded
in Epon-812 after having stained them with 1% toluindine
blue. The lobular mass was divided into three zones of
equal width, extending from the central vein to the portal
area (Glisson’s sheath), namely, pericentral, intermediate,
and periportal zones. To measure the area of vitamin
A-storing lipid droplets in each HSC, and to calculate HSC
density (i.e., cell number per unit area of the liver), we
scanned printed electron micrographs of the cells with an
image scanner (Epson GT-7000S) connected to a personal
computer system (Power Macintosh G3400; Apple Computer). Each area was recognized with Adobe Photoshop
5.0J software, measured, and calculated with NIH Image
1.61. The HSCs, which were recognized by their cell bodies, including their nucleus, were counted in the electron
micrographs.
Fluorescence Microscopy
To detect the autofluorescence of vitamin A, we quickly
cut parts of the excised livers into slices (30 mm ⫻ 30
mm ⫻ 5 mm) and immersed them in 3.7% formaldehyde
for 24 hr at 4°C in total darkness. After 20 ␮m thick
sections had been made with a freezing microtome, they
were examined with a Zeiss Axioskop 20 FL (excitation
filter BP365/12, barrier filter BP495/40) for the detection
of the rapidly fading green autofluorescence characteristic
of vitamin A.
Immunofluorescence Microscopy
The indirect immunofluorescence method for the detection of desmin and ␣-smooth muscle actin (␣-SMA) was
performed on 5 ␮m thick, Zamboni solution (4% paraformaldehyde and 0.2% picric acid in 0.1 M phosphate buffer,
pH 7.4)-fixed, OTC compound (Sakura Finetechnical, Tokyo, Japan)-embedded frozen sections of the livers. The
sections were rinsed three times in 10 mM phosphatebuffered saline, pH 7.4 (PBS), for 5 min. After having been
blocked with 1% bovine serum albumin (BSA) in PBS for
30 min, the sections were incubated for 60 min with mouse
anti-desmin monoclonal antibody (clone D 33; Dako,
Glostrup, Denmark). They were then rinsed in PBS, incubated for 40 min with Alexa 488-labeled rabbit antimouse
IgG antibody previously absorbed with rabbit serum
(Dako), and counterstained with Sytox-orange (Molecular
Probes, Eugene, OR) for 10 min for nucleic acid staining.
Thereafter, the sections were observed under a confocal
laser scanning microscope (LSM 510; Carl Zeiss, Germany). Staining of ␣-SMA was performed by using a
mouse monoclonal antibody (clone 1A4; Dako). After incubation with this antibody for 60 min, the sections were
rinsed in PBS and incubated for 40 min with Alexa 488labeled rabbit anti-mouse IgG antibody previously absorbed with rabbit serum (Dako), counterstained for 10
min with the Sytox-orange, and thereafter observed under
the LSM 510.
Fig. 1. Representative light micrographs of the pericentral zone to the periportal zone in control and
regenerating liver. Orange broken lines show clusters of hepatocytes. Green dotted lines show expanded
Glisson’s sheath. CV, central vein; PV, portal vein; C, control; PHx1, PHx3, PHx5, PHx7, and PHx14 after
70% partial hepatectomy. Scale bar ⫽ 100 ␮m.
902
HIGASHI ET AL.
Statistical Analysis
Statistical analyses were performed by using the oneway analysis of variance (ANOVA). If statistical significance was established, Scheffe’s multiple comparisons test
was used to determine which data sets were significantly
different. P ⬍ 0.05 and ⬍ 0.001 were taken as showing
significance.
RESULTS
General Feature of Liver Lobule
To determine the region to be observed, we first examined the semithin sections stained with 1% toluidine blue.
The liver lobules that clearly show the three zones, i.e.,
pericentral, intermediate, and periportal, were selected
for further analysis by TEM. In the liver on 1 day after
PHx, the arrangement of hepatocyte columns and the
portal area were almost intact (Fig. 1). At 3 and 5 days
after PHx, the hepatocyte columns were shorter and wider
with avascular hepatocyte clusters present in all three
zones (orange dotted lines in Fig. 1), and the thickness of
Glisson’s sheath had become broader (green broken lines
in Fig. 1). By 7 days after PHx, the structure of the liver
had completely returned to normal and remained so at 14
days (Fig. 1).
Morphology of HSCs
HSCs in the intermediate zone of each liver on each day
after PHx were observed in detail. At day 1 after PHx,
most HSCs contained several vitamin A-storing lipid
droplets (⬃ 5), which number was similar to the normal
one (⬃ 6). However, the diameter of the droplets was
smaller (⬃ 1 ␮m) compared with that of normal (⬃ 1.5
␮m). The rough endoplasmic reticulum (rER) was developed within the cytoplasm, and the width of the rER
lumen was slightly larger (⬃ 0.17 ␮m: arrows in Fig. 2,
PHx1) than normal (⬃ 0.13 ␮m: arrows in Fig 2, C). In the
liver 3 days after PHx, the number of lipid droplets (⬃ 3)
was smaller than that at day 1 after PHx, whereas the
diameter of the lipid droplets had not changed, remaining ⬃ 1 ␮m. The rER was well developed within the
cytoplasm, and the width of its lumen was larger (⬃ 0.43
␮m: arrows in Fig. 2, PHx3) than that at day 1 after PHx.
By 5 days after PHx, the number of lipid droplets had
dropped to ⬃ 1, the smallest found in our study. However,
the diameter of the droplets still remained unchanged
compared with that at day 1 after PHx. At this time, the
rER was well developed within the cytoplasm, and the
width of the lumen of rER was the largest (⬃ 0.65 ␮m:
arrows in Fig. 2, PHx5) that we found in the study. In the
liver 7 days after PHx, the number of lipid droplets (⬃ 3)
had increased, but was still smaller than that at day 1
after PHx (⬃ 5) and was equal to that at day 3 post PHx.
The diameter of the lipid droplets had returned to normal.
The width of the rER lumen had narrowed, but was still
Fig. 2. Ultrastructural examination of the intermediate zone in control
and regenerating liver. The hepatic stellate cell (SC) in the liver 5 days
after PHx contains the smallest number of vitamin A-storing lipid droplets (L) found in any of the specimens. Arrows indicate rERs. PC, parenchymal cell; EC, endothelial cell; S, sinusoid; C, control; PHx1, PHx3,
PHx5, PHx7, and PHx14 after 70% partial hepatectomy. Scale bar ⫽ 1
␮m.
903
HEPATIC STELLATE CELLS AND LIVER REGENERATION
TABLE 1. Area of vitamin A-storing lipid droplets and HSC number per unit liver area during
liver regeneration
Days after PHx
Lipid droplet area ␮m2/10,000 ␮m2
Number of HSC/10,000 ␮m2
C (N ⫽ 9)
1 (N ⫽ 9)
3 (N ⫽ 9)
5 (N ⫽ 9)
7 (N ⫽ 9)
14 (N ⫽ 9)
20.6 ⫾ 8.9
1.9 ⫾ 0.1
12.9 ⫾ 7.1
1.8 ⫾ 0.1
11.0 ⫾ 5.2
5.0 ⫾ 0.2**
10.2 ⫾ 5.0
2.8 ⫾ 0.3**
8.9 ⫾ 5.4*
3.2 ⫾ 0.1**
20.5 ⫾ 7.9
2.4 ⫾ 0.1*
Results are expressed as the mean ⫾ SD.
*P ⬍ 0.05, **P ⬍ 0.001 versus C.
C, sham-operated control; N, number of specimens examined.
TABLE 2. Zonal gradient of area of vitamin A-storing lipid droplets during liver regeneration
Days after PHx
␮m2/10,000 ␮m2
Pericentral zone
Intermediate zone
Periportal zone
C (N ⫽ 3)
1 (N ⫽ 3)
3 (N ⫽ 3)
5 (N ⫽ 3)
7 (N ⫽ 3)
14 (N ⫽ 3)
18.5
30.0
13.4
11.3
11.2
9.7
18.1
6.5
8.2
14.9
5.2
7.3
12.4
6.1
3.6
26.2
24.8
14.5
Results are expressed as the median.
C, sham-operated control; N, number of specimens examined.
Fig. 3. Graphic representation of area of vitamin A lipid droplets in HSC (␮m2/cell) showing
vitamin A-storing capacity of HSC in control and
regenerating liver. In the liver 3 days after PHx, the
area of vitamin A lipid droplets in HSC was significantly smaller than that of HSCs in the control
(asterisk, P ⬍ 0.001).
larger (⬃ 0.21 ␮m: arrows in Fig. 2, PHx7) than that at
day 1 after PHx. At the final time point, 14 days after PHx,
the number of lipid droplets (⬃ 9) exceeded normal by ⬃ 3
droplets. The diameter of the lipid droplets (⬃ 1.5 ␮m) and
the width of the lumen of rER (⬃ 0.13 ␮m) were also
normal (arrows in Fig. 2, PHx14).
Morphometry of Vitamin A-Storing Lipid
Droplets
The mean area of vitamin A-storing lipid droplets
within the liver lobule gradually decreased toward day 7
after PHx and then returned to normal within 14 days
after it. The mean area of lipid droplets was the smallest
on day 7 after PHx (8.9 ⫾ 5.4 ␮m2; Table 1). The zonal
heterogeneity of vitamin A-storing lipid droplet area
within each liver lobule, expressed as a symmetrical biphasic distribution with a peak at the intermediate zone
between the portal and central zones of the liver lobule,
disappeared 1 day after PHx (Table 2). The peak zone of
the lipid droplet area within the liver lobule moved from
the intermediate zone to the pericentral zone during the
regeneration period, and the zonal heterogeneity did not
return to normal by 14 days after PHx, even though the
liver volume did return to normal. We also compared the
cell density of HSCs in each zone of the liver lobule at each
sampling after PHx (Table 1). The mean number of HSCs
per 10,000 ␮m2 within the liver lobule gradually increased
toward 3 days after PHx (from the normal of 1.9 ⫾ 0.1 to
5.0 ⫾ 0.2) and then returned to near normal by 14 days
after PHx. No zonal heterogeneity in terms of HSC
number was found (data not shown). The vitamin Astoring capacity of HSCs during liver regeneration was
expressed in terms of area of vitamin A-storing lipid
droplets per HSC (Fig. 3). The value gradually decreased toward 3 days after PHx and returned to the
control level toward 14 days after it. The value at day 3
after PHx was significantly lower (P ⬍ 0.001) than the
control one.
904
HIGASHI ET AL.
Autofluorescence for Vitamin A
Fluorescence micrographs showed vitamin A autofluorescence in the HSCs of the regenerating liver (Fig. 4). The
fluorescing objects in the HSCs often appeared like stars
in the night sky. The intralobular distribution of stored
vitamin A in each liver at each sampling day after PHx
was not inconsistent with the HSCs observed by electron
microscopic morphometry.
Immunofluorescence Microscopy
The number of desmin-positive HSCs increased toward
3 and 5 days after PHx and returned to almost the normal
level 14 days after PHx (Table 3). In the liver 1 day after
PHx, the number of desmin-positive HSCs in the liver
lobules was highest in the periportal zone and decreased
toward the pericentral zone. The same tendency of distribution of these immunopositive HSCs was also observed
in the livers at 5 (Fig. 5), 7, and 14 days after PHx.
However, only in the liver 3 days after PHx were the
desmin-positive HSCs distributed evenly in the three
zones (data not shown). Several ␣-SMA-positive HSCs
were observed in the periportal zone of the liver 3 and 5
(Fig. 5) days after PHx (Table 3). However, none was
observed in the other two zones 3 and 5 days after PHx or
in any of the three zones 1, 7, and 14 days after PHx,
DISCUSSION
This study resulted in two major findings. First, during
liver regeneration, HSCs have the capacity for storing
vitamin A, although it is very low compared with that
under the normal condition. Second, gradient of vitamin
A-storing capacity in HSCs within the hepatic lobules did
not return to normal by 14 days after PHx, even though
the liver volume had done so.
The amount of vitamin A in the regenerating rat liver
was reported earlier (Hauswirth, 1987); however, under
such condition, how the HSCs store the vitamin A has not
yet been revealed. In general, HSCs show remarkable cell
growth when they are activated and lose their stored
vitamin A (Senoo et al., 1984, 1998; Li and Friedman,
2001). Here we showed that during liver regeneration, the
HSCs surely stored vitamin A while showing remarkable
cell growth, although the amount of the stored vitamin
was lower than that under the normal condition. The
results of our fluorescence microscopy study also agree
with these findings.
Following 70% partial hepatectomy in rodents, liver
mass is almost completely restored after 14 days. Hepatocyte proliferation stars after 24 hr in the areas surrounding portal tracts and proceeds to the pericentral
areas by 36 – 48 hr (Michalopoulos and DeFrances, 1997).
As a result of the early hepatocyte proliferation, avascular
clusters consisting of 8 –10 hepatocytes are observed from
3 days after PHx (Martinez-Hernandez et al., 1991), the
formation of which is associated with loss of preexisting
Fig. 4. Detection of vitamin A autofluorescence in HSC in control and
regenerating liver. Vitamin A-associated autofluorescence within each
lobule gradually weaken toward day 7 after PHx and returned to the
control level by 14 days after PHx. CV, central vein; PV, portal vein; C,
control; PHx1, PHx3, PHx5, PHx7, and PHx14 after 70% partial hepatectomy. Scale bar ⫽ 100 ␮m.
905
HEPATIC STELLATE CELLS AND LIVER REGENERATION
TABLE 3. Number of desmin- and ␣-SMA-positive cell per unit liver area during liver regeneration
Days after PHx
number of cells/10,000 ␮m2
C (N ⫽ 9)
1 (N ⫽ 9)
3 (N ⫽ 9)
5 (N ⫽ 9)
7 (N ⫽ 9)
14 (N ⫽ 9)
Desmin
␣-SMA
0.6 ⫾ 0.1
0
0.3 ⫾ 0.1
0
1.6 ⫾ 0.1*
0.4 ⫾ 0.1
1.8 ⫾ 0.5*
0.3 ⫾ 0.1
0.2 ⫾ 0.1
0
0.8 ⫾ 0.2
0
Results are expressed as the mean ⫾ SD. *P ⬍ 0.001 versus C.
C, sham-operated control; N, number of specimens examined.
Fig. 5. Immunofluorescence examination for desmin and ␣-SMA in
pericentral, intermediate, and periportal zones in control liver and in
regenerating liver 5 days after PHx. Liver sections were stained with
monoclonal anti-desmin or ␣-SMA antibody, Alexa-488-labeled second-
ary antibodies in green, and then counterstained with Sytox-Orange for
nuclear staining in red. CV, central vein; PV, portal vein. Scale bar ⫽ 100
␮m.
sinusoidal structure, including the perisinusoidal space in
which HSCs normally reside. Nonparenchymal cells including HSCs enter DNA synthesis 24 hr after hepatocytes, with peak activity at 48 hr or later. Then, HSCs and
sinusoidal endothelial cells proliferate and migrate into
the hepatocyte clusters and restore the normal sinusoidal
architecture. These HSCs that participate in the restoration of normal sinusoidal structure are activated; these
cells usually contain only small amount of vitamin A. As
reported previously (Mabuchi et al., 2004), HSCs play
roles in the hepatic regeneration after PHx by HSC-hepatocyte adhesion.
In the present study, during HSC proliferation, the content of vitamin A in each cell decreases because the vitamin A-lipid droplets also divided into two cells. Thus, two
mechanisms of losing vitamin A from HSCs during hepatic regeneration are suspected: activation of HSCs and
proliferation of HSCs.
The existence of ␣-SMA-positive HSCs 3 and 5 days
after PHx seems to explain partly why HSCs 3 and 5 days
after PHx stored only a small amount of vitamin A in their
cytoplasm, because ␣-SMA is a well-known marker of
activated HSCs (Ramadori et al., 1990). However, we
could not observe ␣-SMA-positive HSCs in livers 1 and 7
days after PHx, although HSCs at these times also showed
a low amount of vitamin A storage. So, more detailed
studies are required to reveal the relationship between
the state of HSCs and their capacity for vitamin A storage.
It was previously reported that the liver lobule physiologically shows zonal heterogeneity in terms of vitamin A
storage (Wake and Sato, 1993; Zou et al., 1998; Higashi
and Senoo, 2003). In our present study, this heterogeneity
of vitamin A storage disappeared during liver regeneration and did not return to normal by 14 days after PHx, at
which time the liver volume generally does return to normal (Michalopoulos and DeFrances, 1997). Our results
obtained by fluorescence microscopy supported this phenomenon. The vitamin A-storing capacity was highest in
the intermediate zone of the three zones in the control
liver, whereas the pericentral zone showed the highest
906
HIGASHI ET AL.
amount of vitamin A storage in the livers 1 to 14 days after
PHx. Regeneration-induced alteration of zonal heterogeneity in the liver lobule with respect to certain enzymes
has also been reported (Anderson et al., 1984). These
alterations might indicate that the proliferating and immature HSCs do not differentiate sufficiently enough
within the liver lobule for storing vitamin A, and that
during liver regeneration, the vitamin A-storing capacity
of HSCs is influenced by the surrounding extracellular
matrix, which also shows a different zonal heterogeneity
in the regenerating liver (Martinez-Hernandez et al.,
1991). In the present study, ␣-SMA-positive HSCs were
localized in the periportal zone of the liver lobule 3 and 5
days after PHx, and this state of activation might partially
be related to the loss of heterogeneity of vitamin A storage
and the delay of recovery. However, the precise mechanisms by which HSCs lose their heterogeneity for vitamin
A storage in the liver lobule are unknown and remain an
open question.
In conclusion, HSCs alter their vitamin A storage capacity during liver regeneration, and the recovery of vitamin A homeostasis requires a much longer time than that
of the liver volume.
ACKNOWLEDGMENTS
The authors thank Dr. Shoichiro Imayoshi (Department
of Cell Biology and Histology, Akita University School of
Medicine) for his valuable discussions. Expert technical
support by Mitsutaka Miura (Department of Cell Biology
and Histology, Akita University School of Medicine) is also
highly appreciated.
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