ARTICLE |
Correspondence to:
Shotaro Sakisaka, Third Department of Medicine, Fukuoka University, Fukuoka, Japan. E-mail: sakisaka@fukuoka-u.ac.jp
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Summary |
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Vascular endothelial growth factor (VEGF) plays a major role in angiogenesis, which is essential for both healing of injured tissue and proliferation of carcinoma cells. In this study we elucidated the expression and role of VEGF in rat liver regeneration after partial hepatectomy. VEGF expression was mainly detected in periportal hepatocytes and reached a maximal level 4872 hr after partial hepatectomy by both immunohistochemistry and in situ hybridization. Similarly, immunohistochemistry for Ki-67 showed that the proliferative activity of sinusoidal endothelial cells was highest in the periportal area and reached a maximal level 72 hr after partial hepatectomy. Moreover, neutralization of VEGF significantly inhibited proliferative activity of hepatocytes (p<0.0001), as well as sinusoidal endothelial cells (p<0.001), at 48 and 96 hr after partial hepatectomy. Conversely, injection of VEGF significantly promoted proliferative activity of hepatocytes (p<0.0001) as well as sinusoidal endothelial cells (p<0.0005) at 48 hr after partial hepatectomy. These results suggest that VEGF promotes proliferation of hepatocytes through reconstruction of liver sinusoids by proliferation of sinusoidal endothelial cells. Furthermore, these data point to a new therapeutic strategy, the use of VEGF and other hepatocyte growth factors in fulminant or severe acute hepatitis. (J Histochem Cytochem 49:121&NDASH;129, 2001)
Key Words: vascular endothelial growth, factor, liver regeneration, partial hepatectomy, sinusoidal endothelial cell, heterogeneity, Ki-67
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Introduction |
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Angiogenesis, the formation of new blood vessels, is a complicated process involving proliferation and migration of endothelial cells. This phenomenon is required for healing of injured tissue, as well as proliferation of carcinoma cells, to supply growth factors, nutrients, and oxygen (
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Materials and Methods |
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Animals
Male Fisher rats weighing 200250 g (Japan SCC; Shizuoka, Japan) were used in all experiments. Rats were kept at a controlled temperature (22C) under a 12-hr lightdark cycle and were maintained on a standard diet and water. All experiments were conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and were approved by the University of Kurume Institutional Animal Care and Use Committee.
Partial Hepatectomy (PH)
Rats underwent 70% PH under light ether anesthesia in the midmorning using the method of
Immunohistochemistry for VEGF
Liver tissues obtained from sacrificed rats were fixed in 10% formalin and embedded in paraffin. Liver tissue sections sliced at 3 µm were placed on silane-coated slides (DAKO; Kyoto, Japan). Sections were deparaffinized with xylene and immersed in absolute and 95% ethanol for 15 sec. The sections were incubated in methanol with 3% (v/v) H2O2 at room temperature (RT) for 10 min to block endogenous peroxidase activity. After incubation, sections were immersed in 85% and 75% ethanol for 15 sec and washed with water. After rehydration in 10 mM PBS with 0.05% Tween-20 (Wako; Osaka, Japan) (T-PBS), sections were preincubated with Protein Block Serum-Free (DAKO) at RT for 30 min. Sections were then incubated with either an anti-VEGF rabbit polyclonal antibody (Santa Cruz Biotechnology; Palo Alto, CA) diluted 1:250 (v/v) with T-PBS or normal rabbit IgG (Inter-Cell Technologies; Hopewell, NJ) diluted 1:2000 with T-PBS at 4C overnight. Sections were washed three times for 5, 10, and 15 min in T-PBS and incubated with anti-rabbit ENVISION PLUS (DAKO) at RT for 60 min. After washing in T-PBS again, sections were incubated in a solution containing 0.1% (w/v) 3-3'-diaminobenzidine-tetrahydrochloride (DAB) and 0.005% (v/v) H2O2 in 0.1 M Tris-HCl buffer (pH 7.6) at RT for 3 min. Nuclear counterstaining was performed with Mayer's hematoxylin for 30 sec.
Immunohistochemistry for Ki-67
Deparaffinized 3-µm sections of liver were immersed in ethanol and incubated to block endogenous peroxidase activity as above. Sections were then autoclaved in 10 mM citrate buffer at 121C for 5 min and cooled slowly to RT. After washing in T-PBS, sections were preincubated with Protein Blocking Serum-Free at RT for 30 min. Immunostaining for Ki-67, a marker for cell proliferation, was performed to evaluate the proliferation of hepatocytes and SECs (
Double Immunostaining for Endothelial Nitric Oxide Synthase and Ki-67
To distinguish SECs from the other nonparenchymal liver sinusoidal cells, SECs were labeled with anti-endothelial nitric oxide synthase (eNOS) mouse monoclonal antibody (
In Situ Hybridization for VEGF
Antisense and sense riboprobes for VEGF were generated by subcloning an Sma ISma-I fragment (380 base pairs) of rat VEGF cDNA into the pGEM3Zf vector (Promega Biotech; Madison, WI) (
For ISH, liver tissues were fixed with 4% paraformaldehyde at 4C for 6 hr. After immersion in 15% sucrose/PBS at 4C for 6 hr, tissues were embedded in Tissue-Tek OCT compound (Miles; Elkhart, IN), and frozen at -75C. Liver tissue sections sliced at 6 µm were placed on silane-coated slides (DAKO). Sections were treated with 1 mg/ml proteinase K (Sigma; St Louis, MO), acetylated with 0.25% acetic anhydride, and incubated with a prehybridization solution containing 50% deionized formamide/2 x SSC at 42C for 1 hr. The hybridization solution contained 0.3 M NaCl, 1 mM EDTA, 10 mM Tris-HCl (pH 7.6), 120 mg/ml herring sperm DNA, 200 mg/ml yeast tRNA, 1 x Denhart's solution, 10% (w/v) dextran sulfate, 50% deionized formamide, and 50100 ng/ml antisense- or sense-labeled riboprobe. After hybridization at 42C for 16 hr, the sections were washed twice with 50% formamide/2 x SSC at 42C for 20 min and treated with RNase A (Boehringer Mannheim) at 37C for 30 min. The sections were washed once with 2 x SSC and twice with 0.1 x SSC at 42C for 30 min. Probes were detected with a sheep polyclonal anti-digoxigenin Fab fragment conjugated to alkaline phosphatase, followed by development in nitroblue tetrazolium and 5-bromo-4-chloro- 3-indolyl phosphate (all from Boehringer Mannheim).
Morphometric Analysis for Cellular Expression of VEGF and Ki-67
We counted the number of hepatocytes positive for VEGF-protein staining and SECs positive for Ki-67 and eNOS staining in the periportal (within 100 µm of the portal area), perivenular (within 100 µm of the central vein), and midzonal areas, and the percentage of positive cells was calculated in each lobular area. The staining intensity for VEGF was evaluated by three grades: strongly positive, weakly positive, and negative.
Injection of Anti-VEGF Antibody or VEGF
To investigate the role of endogenous VEGF in liver regeneration, we studied the effect of anti-VEGF antibody on the proliferation of hepatocytes and SECs before and after the peak time point of Ki-67 expression in SECs. Moreover, we investigated the effect of exogenous VEGF for liver regeneration. In separate experiments, when rats underwent 70% PH, 200 µg/rat of anti-VEGF rabbit IgG (Toagosei; Tsukuba, Japan) (
Statistical Analysis
All data are expressed as means ± SD. Differences between groups were analyzed by the MannWhitney U-test. Comparisons between multiple groups were performed by one-way ANOVA, followed by Fisher's protected least significant difference post-hoc test. p<0.05 was considered statistically significant.
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Results |
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IHC and ISH for VEGF
In the IHC study, VEGF was detected in a few hepatocytes immediately after PH. At 48 hr after PH, VEGF was mainly detected in periportal hepatocytes and less commonly in perivenular hepatocytes (Fig 1A 1C). ISH demonstrated VEGF mRNA mainly in periportal and perivenular hepatocytes (Fig 1E1G). Staining was absent when normal rabbit IgG was substituted for the primary antibody or when sense probe was substituted for antisense probe as a control (Fig 1D and Fig 1H).
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Morphometric Analysis for VEGF-expressing Hepatocytes
The percentage of VEGF-expressing hepatocytes progressively increased and reached maximal levels in periportal and perivenular areas 4872 hr after PH (Fig 2A and Fig 2B). The percentage of hepatocytes strongly expressing VEGF was significantly higher in periportal areas than in perivenular areas (p<0.005).
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Immunohistochemistry for Ki-67 and eNOS
Immediately after PH, Ki-67 was detected in a few hepatocytes and non-parenchymal cells in the liver. At 48 hr after PH, Ki-67 was mainly detected in periportal and midzonal hepatocytes and in some nonparenchymal cells. Endothelial NOS was detected in SECs and the other endothelial cells (Fig 3A and Fig 3B). Staining was absent when normal mouse serum was substituted for the primary antibody (data not shown).
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Morphometric Analysis for Ki-67-expressing SECs
The percentage of Ki-67-expressing SECs was progressively increased after 24 hr and reached maximal levels 72 hr after PH in all three areas. At 72 hr after PH, the percentage of Ki-67-expressing SECs in the periportal area was significantly higher than in the perivenular or midzonal areas (Fig 4).
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Effects of Anti-VEGF Antibody and VEGF on Proliferative Activity of SECs and Hepatocytes
The percentage of Ki-67-expressing SECs 48 hr after PH was significantly lower in anti-VEGF antibody-treated rats and, conversely, was higher in VEGF-treated rats than in untreated rats or control rabbit IgG-treated rats, respectively (p<0.001 and p<0.0001) (Fig 5A). Similarly, at 96 hr after PH, the percentage of Ki-67-expressing SECs was significantly lower in anti-VEGF antibody-treated rats (p<0.0005) (Fig 5B). The percentage of Ki-67-expressing hepatocytes 48 hr after PH was significantly lower in anti-VEGF antibody-treated rats and, conversely, was higher in VEGF-treated rats than in untreated rats or control rabbit IgG-treated rats, respectively (p<0.0001 and p<0.0005) (Fig 5C). Similarly, at 96 hr after PH, the percentage of Ki-67-expressing hepatocytes was significantly lower in anti-VEGF antibody-treated rats (p<0.0005) (Fig 5D). The decreased percentage of Ki-67-expressing SECs in anti-VEGF antibody-treated rats compared with untreated rats was almost similar at 48 and 96 hr after PH. However, the decreased percentage of Ki-67-expressing hepatocytes was higher at 96 hr than at 48 hr after PH.
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Discussion |
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This study is the first to demonstrate that VEGF expression in regenerating rat liver occurs predominantly in periportal hepatocytes. In addition, IHC staining for Ki-67 demonstrated cell proliferation of both SECs and hepatocytes after PH. Finally, anti-VEGF rabbit IgG reduced and, conversely, VEGF promoted the proliferative activity of hepatocytes as well as SECs. These results suggest that VEGF plays a significant role in promoting rat liver regeneration.
Although a variety of growth factors and cytokines have been implicated in liver regeneration, the contribution of VEGF is unclear in detail. Recently, it has been reported that VEGF is a potent angiogenic factor (
Previous studies of VEGF expression in liver homogenates analyzed by Northern blotting, Western blotting, or RT-PCR cannot exclude VEGF production by contaminating platelets (
In the present study, VEGF expression was strongly observed in periportal hepatocytes. Previous studies and our results show that hepatocyte proliferation is more active in periportal areas with lobular heterogeneity after PH () (
VEGF expression was less in perivenular hepatocytes. Although the percentage of VEGF-expressing hepatocytes in both periportal and perivenular areas reached maximal levels 4872 hr after PH, the percentage of VEGF-expressing hepatocytes in perivenular areas was rapidly increased at 2448 hr and was even higher 96 hr after PH compared with periportal areas. Therefore, VEGF expression in perivenular hepatocytes may be affected by other factors in addition to PH. It is possible that a relatively hypoxic condition occurs in the regenerating liver because perivenular hepatocytes are susceptible to hypoxia, which is known to induce VEGF (
VEGF mRNA expression detected by ISH appeared more prominent than VEGF expression detected by IHC. The difference may be attributable to the following possibilities. First, because VEGF is a secreted protein, it may be immediately released from the producing cells. Second, the sensitivity of a VEGF riboprobe against VEGF mRNA may be higher than that of an anti-VEGF antibody against VEGF.
A recent study has reported that nonparenchymal cells expressed VEGF in regenerating liver after PH (
In our results, although anti-VEGF antibody similarly inhibited the proliferative activity of SEC 48 and 96 hr after PH, the effect of anti-VEGF antibody for the proliferative activity of hepatocytes was stronger at 96 hr than at 48 hr after PH. These results indicated that anti-VEGF antibody inhibited significantly but partially the proliferative activity of hepatocytes 48 hr after PH. These results can be interpreted as follows. In the early phase, hepatocytes proliferate at random significantly from periportal areas but rarely accompanying reconstruction of the architecture of hepatic sinusoids. Subsequently, proliferative hepatocytes in ischemic conditions express VEGF to obtain sufficient blood flow. Then hepatocytes proliferate with sufficient blood supplied by accompanying reconstruction of the architecture of hepatic sinusoids. It is likely that hepatocyte proliferation requires growth factors that directly stimulate hepatocytes, such as HGF and TGF-, in the early phase, and growth factors that indirectly stimulate hepatocytes, such as VEGF, in the late phase.
Fulminant hepatitis is associated with profound liver regeneration when patients recover. Our previous report showed that patients with fulminant hepatitis had serum VEGF levels significantly lower than those of healthy individuals, and that serum VEGF levels in survivors of fulminant hepatitis were significantly increased in the recovery phase compared with levels on admission (
In conclusion, we demonstrated that VEGF is produced predominantly by periportal hepatocytes and that it stimulates the proliferative activity of SECs. Furthermore, angiogenesis, as mediated by the proliferation of SECs induced by VEGF, may play a significant role in the proliferation of hepatocytes after PH. Administration of VEGF could open a new therapeutic approach to promote liver regeneration after PH or liver injury and warrants further investigation.
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Acknowledgments |
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We thank Hironori Koga, Motoaki Kin, Takumi Kawaguchi, Atsuko Goto, and Kaori Maeda for technical assistance.
Received for publication February 2, 2000; accepted August 23, 2000.
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