Journal of Histochemistry and Cytochemistry, Vol. 49, 121-130, January 2001, Copyright © 2001, The Histochemical Society, Inc.


ARTICLE

Expression and Role of Vascular Endothelial Growth Factor in Liver Regeneration After Partial Hepatectomy in Rats

Eitaro Taniguchia, Shotaro Sakisakaa, Katsuhiko Matsuob, Kyuichi Tanikawac, and Michio Sataa
a Second Department of Medicine and Kurume University Research Center for Innovative Cancer Therapy, Kurume University School of Medicine, Kurume, Japan
b Bioscience Research Department, Tsukuba Research Laboratory, Toagosei Co. Ltd., Tsukuba, Japan
c International Institute for Liver Research, Kurume, Japan

Correspondence to: Shotaro Sakisaka, Third Department of Medicine, Fukuoka University, Fukuoka, Japan. E-mail: sakisaka@fukuoka-u.ac.jp or eitaro@med.kurume-u.ac.jp


  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 48–72 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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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 (Algire et al. 1945 ; Folkman 1995a , Folkman 1995b ). Various angiogenic factors have been identified, but vascular endothelial growth factor (VEGF) is the most potent and specific growth factor for both angiogenesis and vasculogenesis (Mustonen and Alitalo 1995 ; Shibuya 1995 ). VEGF has been demonstrated to play a central role in division, migration, and tubule formation of vascular endothelial cells through its receptors, kinase insert domain-containing receptor/fetal liver kinase 1 (KDR/Flk-1) and fms-like tyrosine kinase 1 (Flt-1) (Mustonen and Alitalo 1995 ; Shibuya 1995 ; Brown et al. 1997 ; Ferrara and Davis-Smyth 1997 ). Proliferation of sinusoidal endothelial cells (SECs) is predominantly regulated by VEGF (Yamane et al. 1994 ), although other angiogenic factors such as platelet-derived growth factor and fibroblast growth factor also participate in this process (Folkman and Klagsbrun 1987 ; Gospodarowicz et al. 1987 ; Leveen et al. 1994 ; Folkman 1995a , Folkman 1995b ; Shibuya 1995 ).In liver regeneration after partial hepatectomy (PH), both hepatocytes and nonparenchymal cells express VEGF mRNA (Mochida et al. 1996 ), suggesting that VEGF plays a significant role in liver regeneration. However, the expression and role of VEGF have not been examined in detail in liver regeneration. In this study we investigated the expression of VEGF in regenerating rat liver after PH by in situ hybridization (ISH) and immunohistochemistry (IHC). Furthermore, we elucidated the role of endogenous VEGF by its neutralization and subsequently assessing proliferative activity of SECs and hepatocytes. We found that VEGF was mainly expressed in periportal hepatocytes and we demonstrated that VEGF was significantly involved in proliferation of hepatocytes associated with proliferation of SECs after PH in rats.


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Animals
Male Fisher rats weighing 200–250 g (Japan SCC; Shizuoka, Japan) were used in all experiments. Rats were kept at a controlled temperature (22C) under a 12-hr light–dark 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 Higgins and Anderson 1931 . Briefly, the liver was exposed through a 1–2-cm midline abdominal incision and the two anterior lobes were exteriorized, the vascular pedicles were ligated, and the lobes were excised. Rats were sacrificed at 0, 12, 24, 48, 72, 96, or 168 hr after PH. Numbers of rats used in each experiment are indicated in the figure legends.

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 (Gerdes et al. 1983 , Gerdes et al. 1984 ). Sections were incubated with either an anti-Ki-67 mouse monoclonal antibody (Immunotech; Marseille, France) diluted 1:100 (v/v) with T-PBS or normal mouse serum diluted 1:2000 with T-PBS at 4C overnight. Sections were washed in T-PBS and incubated with a biotinylated anti-mouse IgG (Vectastain ABC Kit; Vector Labs, Burlingame, CA) diluted 1:200 (v/v) with T-PBS at RT for 30 min. After washing in T-PBS, sections were incubated with avidin–biotin–peroxidase complex (Vectastain ABC Kit) at RT for 30 min. Sections were then incubated in DAB and H2O2 as above. Nuclear counterstaining was performed with Mayer's hematoxylin for 10 min.

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 (Rockey and Chung 1998 ). Deparaffinized 3-m 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 as above and then preincubated with Protein Blocking Serum-Free for 30 min. Sections were incubated with anti-eNOS mouse monoclonal antibody (Transduction Laboratories; Lexington, KY) and anti-Ki-67 mouse monoclonal antibody (Immunotech) diluted 1:100 (v/v) with T-PBS at 4C overnight. Sections were washed in T-PBS and incubated with a biotinylated anti-mouse IgG (Vectastain ABC Kit) diluted 1:200 (v/v) with T-PBS at RT for 30 min. After washing in T-PBS, sections were incubated with avidin–biotin–peroxidase complex (Vectastain ABC Kit) at RT for 30 min. Sections were then incubated in DAB and H2O2 as above. Nuclear counterstaining was performed with Mayer's hematoxylin for 10 min.

In Situ Hybridization for VEGF
Antisense and sense riboprobes for VEGF were generated by subcloning an Sma I–Sma-I fragment (380 base pairs) of rat VEGF cDNA into the pGEM3Zf vector (Promega Biotech; Madison, WI) (Keck et al. 1989 ). The plasmids were used to generate digoxigenin-labeled antisense and sense riboprobes by T7 RNA polymerase using an RNA labeling kit (Boehringer Mannheim; Mannheim, Germany).

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 50–100 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) (Asano et al. 1995 ) or normal rabbit IgG (Inter-Cell Technologies) was administered via the tail vein. These rats were sacrificed 48 or 96 hr after PH. Similarly, 500 ng/rat of VEGF (Toagosei) was administered 0 and 24 hr after PH. These rats were sacrificed 48 hr after PH. Numbers of rats used in each experiment are indicated in the figure legends.

Statistical Analysis
All data are expressed as means ± SD. Differences between groups were analyzed by the Mann–Whitney 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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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 1E–1G). 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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Figure 1. Expression of VEGF and VEGF mRNA in livers after PH. Immunohistochemical staining of VEGF in the liver lobules (A), periportal (B) and perivenular (C) areas, and ISH of VEGF mRNA in the liver lobules (E), periportal (F) and perivenular (G) areas. VEGF was detected in periportal (arrows) and perivenular (arrowheads) hepatocytes. Staining was absent with normal rabbit serum (D) or with sense probe (H). p, portal vein; c, central vein. Original magnifications: A,E x 150; B–D,F–H x 400.

Morphometric Analysis for VEGF-expressing Hepatocytes
The percentage of VEGF-expressing hepatocytes progressively increased and reached maximal levels in periportal and perivenular areas 48–72 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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Figure 2. Morphometric analysis for VEGF-expressing hepatocytes in periportal (A) and perivenular (B) areas. Liver sections were obtained from four rats at each time point. The numbers of VEGF-expressing hepatocytes with strongly positive staining (solid) or weakly positive staining (blank) are expressed as percentages. Values are expressed as means ± SD (n = 16 liver lobules in each group). p<0.0001 at 48 or 72 hr vs 0 hr after PH.

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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Figure 3. Immunohistochemical staining of Ki-67 and eNOS in livers after PH. Ki-67 was detected in the nuclei and eNOS was detected in the cytoplasm. Double-positive cells for Ki-67 and eNOS indicated proliferative endothelial cells. Note Ki-67-positive (arrow) and -negative (arrowheads) SECs (A,B). Liver sections were at 48 hr (A) and 96 hr (B) after PH. p, portal vein; c, central vein. Original magnifications: A x 150; B x 400.

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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Figure 4. Morphometric analysis for Ki-67-expressing SECs. Liver sections were obtained from four rats in each group at each time point. Ki-67-expressing hepatocytes are expressed as a percentage. Values are expressed as means ± SD (n = 16 liver lobules in each group). •, periportal area; {blacksquare}, perivenular area; {blacktriangleup}, midzonal area. *p<0.0001 at 96 hr vs 0 hr after PH in each area. {dagger}p<0.05 for periportal vs perivenular or midzonal areas at 72 hr after PH.

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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Figure 5. Effects of anti-VEGF rabbit IgG, VEGF, or nonimmunoreactive rabbit IgG on Ki-67-labeling index of SECs (A,B) and hepatocytes (C,D) in the periportal area 48 and 96 hr after PH. Anti-VEGF, VEGF, rabbit IgG, and control represent rats treated with anti-VEGF rabbit IgG, VEGF, nonimmunoreactive rabbit IgG, or no treatment, respectively. Injection of anti-VEGF rabbit IgG significantly reduced and, conversely, injection of VEGF significantly promoted the proliferative activity of both SECs and hepatocytes. Liver sections were obtained from four rats in each group. Values are expressed as means ± SD (n = 25 liver lobules in each group) *p<0.0001; {dagger}p<0.0005, {ddagger}p<0.001. NS, not significant.


  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 (Mustonen and Alitalo 1995 ; Shibuya 1995 ), promoting proliferation of carcinoma cells (Shibuya 1995 ; Brown et al. 1997 ; Ferrara and Davis-Smyth 1997 ) and healing of injured tissue (Brown et al. 1997 ; Ferrara and Davis-Smyth 1997 ). We hypothesized that VEGF was required for reconstruction of hepatic sinusoids in the regenerating liver and that VEGF sequentially participates in liver regeneration by facilitating hepatocyte proliferation.

Previous studies of VEGF expression in liver homogenates analyzed by Northern blotting, Western blotting, or RT-PCR cannot exclude VEGF production by contaminating platelets (Banks et al. 1998 ; Wartiovaara et al. 1998 ). Furthermore, these methods are not able to determine the distribution of VEGF-expressing cells in the liver. Therefore, we examined regenerating liver by ISH with antisense probe for VEGF and by IHC with an anti-VEGF antibody to determine VEGF-expressing cells.

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 (Gerlach et al. 1997 ). Recent studies on expression of key growth factors, such as hepatocyte growth factor (HGF) (Ishiki et al. 1992 ; Shiota et al. 1994 ), transforming growth factor-alpha (TGF-{alpha}) (Webber et al. 1993 ; Russell et al. 1996 ), and heparin-binding epidermal growth factor-like growth factor (HB-EGF) (Ito et al. 1994 ; Kiso et al. 1995 ), in hepatocyte proliferation after PH have not shown a correlation between the distribution of these growth factor's expression and lobular heterogeneity of hepatocyte proliferation. We demonstrated a correlative distribution between VEGF-expressing hepatocytes and proliferation of SECs. The lobular heterogeneity of VEGF-expressing hepatocytes suggests that reconstruction of hepatic sinusoids by SECs begins from periportal areas at which hepatocyte proliferation occurs in the regenerating liver. Previous studies have demonstrated that VEGF is expressed in epithelial cells of stomach or skin, and is closely associated with wound healing of tissues (Detmar et al. 1995 ; Frank et al. 1995 ; Takahashi et al. 1997 ), suggesting that VEGF expressed in hepatocytes, epithelial cells in liver, had a role in liver regeneration.

VEGF expression was less in perivenular hepatocytes. Although the percentage of VEGF-expressing hepatocytes in both periportal and perivenular areas reached maximal levels 48–72 hr after PH, the percentage of VEGF-expressing hepatocytes in perivenular areas was rapidly increased at 24–48 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 (Shweiki et al. 1992 ; Brown et al. 1997 ; Ferrara and Davis-Smyth 1997 ).

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 (Mochida et al. 1996 ), whereas VEGF was not detected in nonparenchymal cells by either IHC or ISH in this study. We suggest that nonparenchymal cells, especially Kupffer cells, take up platelets that express VEGF mRNA (Wartiovaara et al. 1998 ), or that isolation of nonparenchymal cells artifactually induces the expression of VEGF in these cells.

Widmann and Fahimi 1975 have demonstrated that SECs increased in number and reached a maximal level 96 hr after PH. Our results demonstrated that the percentage of Ki-67-expressing SECs reached maximal level 72 hr after PH. This discrepancy could be explained by different procedures use to analyze SEC proliferation. Although the previous study examined the number of whole SECs, we examined the proliferative SECs using the well-documented cell proliferation marker Ki-67 (Gerdes et al. 1983 , Gerdes et al. 1984 ). However, proliferation of SECs has been demonstrated only in periportal areas but not in perivenular and midzonal areas. We found that proliferation of SECs was most active in periportal areas, moderately active in perivenular areas, and less active in midzonal areas, suggesting that VEGF produced by periportal and perivenular hepatocytes promoted proliferative activity of SECs, probably in a paracrine fashion. Furthermore, the proliferative activity of periportal SECs was significantly higher 48 and 72 hr after PH, corresponding to increased proliferative activity of periportal hepatocytes.

Assy et al. 1999 have demonstrated that injection of VEGF promoted the proliferative activity of hepatocytes, suggesting that VEGF promoted liver regeneration. However, it is unclear how VEGF promotes the proliferative activity of hepatocytes that do not express any of the VEGF receptors KDR/Flk-1 and Flt-1 (Akira et al. 1994 ; Mochida et al. 1996 ). Moreover, they have not examined proliferative activity of SECs that expressed VEGF receptors (Akira et al. 1994 ; Mochida et al. 1996 ). We first demonstrated that endogenous and exogenous VEGF promoted the proliferation of SECs during liver regeneration after PH and that the reconstruction of hepatic sinusoids appeared to promote the proliferation of hepatocytes. VEGF in transplanted hepatic tissues promotes liver tissue formation accompanied by vascular structures (Ajioka et al. 1999 ). Taken together, these results suggest that VEGF promotes liver tissue formation, accompanied by reconstruction of hepatic sinusoids by SECs, in liver regeneration after PH or liver injury.

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-{alpha}, 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 (Akiyoshi et al. 1998 ), suggesting that VEGF has a critical role in liver regeneration after fulminant hepatitis. Recently, HGF was implicated in liver regeneration after PH (Sakon et al. 1992 ; Tomiya et al. 1992 ) or liver injury (Tsubouchi et al. 1992 ; Shiota et al. 1995 ) in humans, and recombinant HGF has been suggested as a therapeutic application for liver diseases that require regeneration (Ishiki et al. 1992 ; Kaibori et al. 1997 ; Patijn et al. 1998 ). For future clinical applications, exogenous VEGF may provide a new therapeutic means for liver regeneration after PH or liver injury, such as fulminant hepatitis by itself or in combination with HGF.

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.


  Acknowledgments

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.


  Literature Cited
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Summary
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Materials and Methods
Results
Discussion
Literature Cited

Ajioka I, Akaike T, Watanabe Y (1999) Expression of vascular endothelial growth factor promotes colonization, vascularization, and growth of transplanted hepatic tissues in the mouse. Hepatology 29:396-402[Medline]

Akira Y, Lata S, Sachiko Y, Noriko G, Tomoko T, Gera N, Masabumi S (1994) A new communication system between hepatocytes and sinusoidal endothelial cells in liver through vascular endothelial growth factor and Flt tyrosine kinase receptor family (Flt-1 and KDR/Flk-1). Oncogene 9:2683-2690[Medline]

Akiyoshi F, Sata M, Suzuki H, Uchimura Y, Mitsuyama K, Matsuo K, Tanikawa K (1998) Serum vascular endothelial growth factor levels in various liver diseases. Dig Dis Sci 43:41-45[Medline]

Algire GH, Chalkley HW, Legallais FY, Park HD (1945) Vascular reactions of normal and malignant tissues in vivo: 1. Vascular reactions of mice to wounds and to normal and neoplastic transplants. J Natl Cancer Inst 6:73-85

Asano M, Yukita A, Matsumoto T, Matsuo K, Kondo S, Suzuki H (1995) Isolation and characterization of neutralizing monoclonal antibodies to human vascular endothelial growth factor/vascular permeability factor121 (VEGF/VPF121). Hybridoma 14:475-480[Medline]

Assy N, Spira G, Paizi M, Shenkar L, Kraizer Y, Cohen T, Neufeld G, Dabbah B, Enat R, Baruch Y (1999) Effect of vascular endothelial growth factor on hepatic regenerative activity following partial hepatectomy in rats. J Hepatol 30:911-915[Medline]

Banks RE, Forbes MA, Kinsey SE, Stanley A, Ingham E, Walters C, Selby PJ (1998) Release of the angiogenic cytokine vascular endothelial growth factor (VEGF) from platelets: significance for VEGF measurements and cancer biology. Br J Cancer 77:956-964[Medline]

Brown LF, Detmar M, Claffey K, Nagy JA, Feng D, Dvorak AM, Dvorak HF (1997) Vascular permeability factor/vascular endothelial growth factor: a multifunctional angiogenic cytokine. EXS 79:233-269[Medline]

Detmar M, Yeo KT, Nagy JA, Van de Water L, Brown LF, Berse B, Elicker BM, Ledbetter S, Dvorak HF (1995) Keratinocyte-derived vascular permeability factor (vascular endothelial growth factor) is a potent mitogen for dermal microvascular endothelial cells. J Invest Dermatol 105:44-50[Abstract]

Ferrara N, Davis-Smyth T (1997) The biology of vascular endothelial growth factor. Endocrine Rev 18:4-25[Abstract/Free Full Text]

Folkman J (1995a) Angiogenesis in cancer, vascular, rheumatoid and other disease. Nature Med 1:27-31[Medline]

Folkman J (1995b) Clinical applications of research on angiogenesis. N Engl J Med 333:1757-1763[Free Full Text]

Folkman J, Klagsbrun M (1987) Angiogenic factors. Science 235:442-447[Medline]

Frank S, Hubner G, Breier G, Longaker MT, Greenhalgh DG, Werner S (1995) Regulation of vascular endothelial growth factor expression in cultured keratinocytes. Implications for normal and impaired wound healing. J Biol Chem 270:12607-12613[Abstract/Free Full Text]

Gerdes J, Lemke H, Baisch H, Wacker HH, Schwab U, Stein H (1984) Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67. J Immunol 133:1710-1715[Abstract/Free Full Text]

Gerdes J, Schwab U, Lemke H, Stein H (1983) Production of a mouse monoclonal antibody reactive with a human nuclear antigen associated with cell proliferation. Int J Cancer 31:13-20[Medline]

Gerlach C, Sakkab DY, Scholzen T, Dassler R, Alison MR, Gerdes J (1997) Ki-67 expression during rat liver regeneration after partial hepatectomy. Hepatology 26:573-578[Medline]

Gospodarowicz D, Ferrara N, Schweigerer L, Neufeld G (1987) Structural characterization and biological functions of fibroblast growth factor. Endocrine Rev 8:95-114[Medline]

Higgins GM, Anderson RM (1931) Experimental pathology of the liver: 1. Restoration of the liver of the white rat following partial surgical removal. Arch Pathol 12:186-202

Ishiki Y, Ohnishi H, Muto Y, Matsumoto K, Nakamura T (1992) Direct evidence that hepatocyte growth factor is a hepatotrophic factor for liver regeneration and has a potent antihepatitis effect in vivo. Hepatology 16:1227-1235[Medline]

Ito N, Kawata S, Tamura S, Kiso S, Tsushima H, Damm D, Abraham JA, Higashiyama S, Taniguchi N, Matsuzawa Y (1994) Heparin-binding EGF-like growth factor is a potent mitogen for rat hepatocytes. Biochem Biophys Res Commun 198:25-31[Medline]

Kaibori M, Kwon A, Nakagawa M, Wei T, Uetsuji S, Kamiyama Y, Okumura T, Kitamura N (1997) Stimulation of liver regeneration and function after partial hepatectomy in cirrhotic rats by continuous infusion of recombinant human hepatocyte growth factor. J Hepatol 27:381-390[Medline]

Keck PJ, Hauser SD, Krivi G, Sanzo K, Warren T, Feder J, Connolly DT (1989) Vascular permeability factor, an endothelial cell mitogen related to PDGF. Science 246:1309-1312[Medline]

Kiso S, Kawata S, Tamura S, Higashiyama S, Ito N, Tsushima H, Taniguchi N, Matsuzawa Y (1995) Role of heparin-binding epidermal growth factor-like growth factor as a hepatotrophic factor in rat liver regeneration after partial hepatectomy. Hepatology 22:1584-1590[Medline]

Leveen P, Pekny M, Gebre–Medhin S, Swolin B, Larsson E, Betsholtz C (1994) Mice deficient for PDGF B show renal, cardiovascular, and hematological abnormalities. Genes Dev 8:1875-1887[Abstract]

Mochida S, Ishikawa K, Inao M, Shibuya M, Fujiwara K (1996) Increased expressions of vascular endothelial growth factor and its receptors, flt-1 and KDR/flk-1, in regenerating rat liver. Biochem Biophys Res Commun 226:176-179[Medline]

Mustonen T, Alitalo K (1995) Endothelial receptor tyrosine kinases involved in angiogenesis. J Cell Biol 129:895-898[Medline]

Patijn GA, Lieber A, Schowalter DB, Schwall R, Kay MA (1998) Hepatocyte growth factor induces hepatocyte proliferation in vivo and allows for efficient retroviral-mediated gene transfer in mice. Hepatology 28:707-716[Medline]

Rockey DC, Chung JJ (1998) Reduced nitric oxide production by endothelial cells in cirrhotic rat liver: endothelial dysfunction in portal hypertension. Gastroenterology 114:344-351[Medline]

Russell WE, Kaufmann WK, Sitaric S, Luetteke NC, Lee DC (1996) Liver regeneration and hepatocarcinogenesis in transforming growth factor-alpha-targeted mice. Mol Carcinogen 15:183-189[Medline]

Sakon M, Monden M, Gotoh M, Kanai T, Umeshita K, Mori T, Tsubouchi H, Daikuhara Y (1992) Hepatocyte growth factor concentrations after liver resection. Lancet 339:818[Medline]

Shibuya M (1995) Role of VEGF-flt receptor system in normal and tumor angiogenesis. Adv Cancer Res 67:281-316[Medline]

Shiota G, Okano J, Kawasaki H, Kawamoto T, Nakamura T (1995) Serum hepatocyte growth factor levels in liver diseases: clinical implications. Hepatology 21:106-112[Medline]

Shiota G, Wang TC, Nakamura T, Schmidt EV (1994) Hepatocyte growth factor in transgenic mice: effects on hepatocyte growth, liver regeneration and gene expression. Hepatology 19:962-972[Medline]

Shweiki D, Itin A, Soffer D, Keshet E (1992) Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis. Nature 359:843-845[Medline]

Takahashi M, Kawabe T, Ogura K, Maeda S, Mikami Y, Kaneko N, Terano A, Omata M (1997) Expression of vascular endothelial growth factor at the human gastric ulcer margin and in cultured gastric fibroblasts: a new angiogenic factor for gastric ulcer healing. Biochem Biophys Res Commun 234:493-498[Medline]

Tomiya T, Tani M, Yamada S, Hayashi S, Umeda N, Fujiwara K (1992) Serum hepatocyte growth factor levels in hepatectomized and nonhepatectomized surgical patients. Gastroenterology 103:1621-1624[Medline]

Tsubouchi H, Kawakami S, Hirono S, Miyazaki H, Kimoto M, Arima T, Sekiyama K, Yoshiba M, Arakaki N, Daikuhara Y (1992) Prediction of outcome in fulminant hepatic failure by serum human hepatocyte growth factor. Lancet 340:307[Medline]

Wartiovaara U, Salven P, Mikkola H, Lassila R, Kaukonen J, Joukov V, Orpana A, Ristimaki A, Heikinheimo M, Joensuu H, Alitalo K, Palotie A (1998) Peripheral blood platelets express VEGF-C and VEGF which are released during platelet activation. Thromb Haemost 80:171-175[Medline]

Webber EM, FitzGerald MJ, Brown PI, Bartlett MH, Fausto N (1993) Transforming growth factor-alpha expression during liver regeneration after partial hepatectomy and toxic injury, and potential interactions between transforming growth factor-alpha and hepatocyte growth factor. Hepatology 18:1422-1431[Medline]

Widmann JJ, Fahimi HD (1975) Proliferation of mononuclear phagocytes (Kupffer cells) and endothelial cells in regenerating rat liver. Am J Pathol 80:349-366[Abstract]

Yamane A, Seetharam L, Yamaguchi S, Gotoh N, Takahashi T, Neufeld G, Shibuya M (1994) A new communication system between hepatocytes and sinusoidal endothelial cells in liver through vascular endothelial growth factor and Flt tyrosine kinase receptor family (Flt-1 and KDR/Flk-1). Oncogene 9:2683-2690[Medline]