Vitamin A storage in hepatic stellate cells in the regenerating rat liver: With special reference to zonal heterogeneity
Article first published online: 7 AUG 2005
Copyright © 2005 Wiley-Liss, Inc.
The Anatomical Record Part A: Discoveries in Molecular, Cellular, and Evolutionary Biology
Volume 286A, Issue 2, pages 899–907, October 2005
How to Cite
Higashi, N., Sato, M., Kojima, N., Irie, T., Kawamura, K., Mabuchi, A. and Senoo, H. (2005), Vitamin A storage in hepatic stellate cells in the regenerating rat liver: With special reference to zonal heterogeneity. Anat. Rec., 286A: 899–907. doi: 10.1002/ar.a.20230
- Issue published online: 21 SEP 2005
- Article first published online: 7 AUG 2005
- Manuscript Accepted: 4 JUN 2005
- Manuscript Received: 27 FEB 2005
- Ministry of Education, Culture, Sports, Science, and Technology of Japan. Grant Number: B 16790118
- hepatic stellate cell;
- vitamin A;
- liver regeneration;
- partial hepatectomy;
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 α-smooth muscle actin (α-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.
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 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 hepatectomy (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
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.
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).
Seventy percent partial hepatectomy was performed by using a modification of the technique described by Higgins and Anderson (1931). Control rats underwent a sham-operation as mentioned above.
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, fluorescence microscopy, and immunofluorescence microscopy, we took specimens by systematic random sampling. This sampling method minimized the measurement influences.
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.
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 μm 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.
The indirect immunofluorescence method for the detection of desmin and α-smooth muscle actin (α-SMA) was performed on 5 μm 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 phosphate-buffered 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 α-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 488-labeled 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.
Statistical analyses were performed by using the one-way 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.
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 μm) compared with that of normal (∼ 1.5 μm). The rough endoplasmic reticulum (rER) was developed within the cytoplasm, and the width of the rER lumen was slightly larger (∼ 0.17 μm: arrows in Fig. 2, PHx1) than normal (∼ 0.13 μm: 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 μm. The rER was well developed within the cytoplasm, and the width of its lumen was larger (∼ 0.43 μm: 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 μm: 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 larger (∼ 0.21 μm: 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 μm) and the width of the lumen of rER (∼ 0.13 μm) 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 μm2; 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 μm2 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 A-storing 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.
|Days after PHx|
|C (N = 9)||1 (N = 9)||3 (N = 9)||5 (N = 9)||7 (N = 9)||14 (N = 9)|
|Lipid droplet area μm2/10,000 μm2||20.6 ± 8.9||12.9 ± 7.1||11.0 ± 5.2||10.2 ± 5.0||8.9 ± 5.4*||20.5 ± 7.9|
|Number of HSC/10,000 μm2||1.9 ± 0.1||1.8 ± 0.1||5.0 ± 0.2**||2.8 ± 0.3**||3.2 ± 0.1**||2.4 ± 0.1*|
|μm2/10,000 μm2||Days after PHx|
|C (N = 3)||1 (N = 3)||3 (N = 3)||5 (N = 3)||7 (N = 3)||14 (N = 3)|
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.
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 α-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,
|number of cells/10,000 μm2||Days after PHx|
|C (N = 9)||1 (N = 9)||3 (N = 9)||5 (N = 9)||7 (N = 9)||14 (N = 9)|
|Desmin||0.6 ± 0.1||0.3 ± 0.1||1.6 ± 0.1*||1.8 ± 0.5*||0.2 ± 0.1||0.8 ± 0.2|
|α-SMA||0||0||0.4 ± 0.1||0.3 ± 0.1||0||0|
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 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 α-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 α-SMA is a well-known marker of activated HSCs (Ramadori et al., 1990). However, we could not observe α-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 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, α-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.
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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