Clinical Cancer Research Targets Frontiers in Basic Cancer Research
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hisai, H.
Right arrow Articles by Niitsu, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hisai, H.
Right arrow Articles by Niitsu, Y.
Clinical Cancer Research Vol. 9, 4852-4859, October 15, 2003
© 2003 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

Increased Expression of Angiogenin in Hepatocellular Carcinoma in Correlation with Tumor Vascularity

Hiroyuki Hisai, Junji Kato1, Masayoshi Kobune, Tsuzuku Murakami, Koji Miyanishi, Minoru Takahashi, Naohito Yoshizaki, Rishu Takimoto, Takeshi Terui and Yoshiro Niitsu

Fourth Department of Internal Medicine, Sapporo Medical University School of Medicine, Sapporo 060-8543, Japan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: Neovascularization is known to be one of the major characteristics of human hepatocellular carcinoma (HCC). Angiogenin (ANG), originally discovered in a human colon cancer cell line, is a liver-derived polypeptide that shows strong angiogenic activity in vivo. However, the role of ANG on the development of HCC remains unknown. The present study was designed to examine the implication of ANG in the neovascularization of human HCC.

Experimental Design: Forty-one HCC patients who had undergone conventional celiac angiography were used in this study. ANG protein expression and microvessel density (MVD) in HCC specimens obtained by liver biopsy or surgical resection were examined by immunohistochemistry, and the levels were quantified by the KS-400 image analyzing system. ANG mRNA expression in liver tissues was evaluated by in situ hybridization. Serum ANG concentrations were measured by an ELISA. Survival rates were calculated using the Kaplan-Meier method.

Results: Immunohistochemistry and in situ hybridization showed greater increments of ANG protein expression and mRNA expression, respectively, in HCC tissues than in the surrounding nontumorous tissues. MVD within tumorous tissues increased according to dedifferentiation of the histological grade of HCC, showing a significant correlation (r = 0.877, P = 0.0009) with ANG expression levels. Mean ± SD serum ANG levels of healthy subjects and chronic hepatitis (CH) patients were 362.3 ± 84.1 ng/ml and 331.9 ± 133.8 ng/ml, respectively, with no significant difference. Serum ANG levels of liver cirrhosis patients (242.4 ± 126.9 ng/ml) were lower than those of healthy subjects or CH patients and decreased as the fibrosis grade advanced. In HCC patients, despite the cirrhotic background, serum ANG levels increased as the tumor vascularity increased (197.8 ± 64.9 ng/ml for hypovascular, 326.7 ± 148.6 ng/ml for hypervascular, and 405.0 ± 121.3 ng/ml for very hypervascular), in good accordance with histological grading, and significantly decreased (P = 0.015) after successful treatment with transcatheter arterial embolization or percutaneous ethanol injection. HCC patients were conventionally divided into two groups according to the serum level of ANG, those with values higher than the mean level (332.9 ± 143.8 ng/ml) and those with values lower than the mean,; the 5-year survival rate of the latter group was determined to be significantly higher than that in the former group.

Conclusions: These results suggest that ANG is one of the neovascularization defining factors of HCC. Thus, measuring serum ANG may assist in monitoring the disease, and targeting ANG may provide a new strategy for treating advanced HCC.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
HCC2 is a highly invasive tumor characterized by vigorous neovascularization that is usually detectable by routine diagnostic imaging modalities such as contrast-enhanced computed tomography and celiac angiography (1 , 2) . Recent studies have suggested that some angiogenic factors such as VEGF, platelet-derived endothelial growth factor (PD-ECGF), basic fibroblast growth factor (bFGF) released from the tumor itself, tumor-infiltrating inflammatory cells, and/or tumor stroma cells, participate in the neovascularization of human HCC. In some previous reports, expressions of these factors in cancerous tissue and serum concentration of these factors have been disclosed to correlate with tumor vascularity and with the survival of HCC patients (3, 4, 5, 6, 7) . In other reports, contrarily, expressions of VEGF and platelet-derived endothelial growth factor in the tumor were found to be correlated with the grade of hepatic fibrosis, but not with the MVD of the tumor (8 , 9) . Furthermore, circulating basic fibroblast growth factor levels in HCC patients were also shown to correlate well with the degree of tumor capsular infiltration but not that of the tumor vascularity (10) .

ANG is a 14.1 kDa polypeptide that regulates angiogenesis under both physiological and pathological conditions (11, 12, 13) . Previous reports have demonstrated that a high expression of ANG in the tumor tissue or elevated serum ANG concentration was observed in patients with various malignancies, including colorectal carcinoma, melanoma, pancreatic carcinoma, and urothelial carcinoma (14, 15, 16, 17, 18, 19) , suggesting its involvement in the neovascularization of various neoplasms. However, it has not been elucidated whether ANG participates in the tumor angiogenesis of human HCC. In the present study, therefore, we elucidated the correlation of neovascularization and ANG expression in human HCC.


   PATIENTS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients.
All of the subjects provided written informed consent for use of serum or liver tissue before the present study. The study enrolled 41 HCC patients (32 men and 9 women; ages, mean ± SD, 63 ± 10 years) who underwent liver biopsy or surgical resection and selective angiography at Sapporo Medical University Hospital, Sapporo National Hospital, or Nikko Memorial Hospital between January 1994 and December 1996 (Table 1)Citation . The diagnosis was made histologically on biopsied or resected specimens. The clinical staging of HCC was determined according to criteria of the International Union against Cancer Stages of HCC (20) . Fifteen patients with CH (7 hepatitis B and 8 hepatitis C; ages, mean ± SD, 53 ± 14 years), 24 patients with LC (10 hepatitis B and 14 hepatitis C; ages, mean ± SD, 65 ± 9 years) and 31 healthy volunteers (ages, mean ± SD, 45 ± 14 years) were also enrolled to measure their serum ANG levels. Serum samples were taken and stored at -70°C until use.


View this table:
[in this window]
[in a new window]

  		Table 1 Clinical characteristics of the 41 patients with HCC

 
Selective Angiography and Evaluation of Tumor Vascularity.
Selective angiography was performed in all of the HCC patients. Iopamidol 370 (Schering, Berlin, Germany) was injected at the rate of 7 ml/s via the celiac artery or 4 ml/s via the right, the left, or the proper hepatic artery, depending on tumor location. The tumor vascularity was assessed by the enhancement effect on the arterial phase of angiography according to the method of Honda et al. with some modification (21) . In brief, tumors that were markedly enhanced either by celiac angiography or by selective angiography were assessed as "very hypervascular" lesions, those minimally to mildly enhanced by celiac angiography but markedly enhanced by selective angiography were assessed as hypervascular, and those not enhanced at all by celiac angiography but slightly enhanced or not enhanced by selective angiography were assessed as hypovascular.

Tissue Samples.
Ultrasound-guided percutaneous needle liver biopsy was performed using Monopty needles (C.R. Bard, Convington, GA). Each fresh biopsy specimen or surgically resected tissue was divided into two portions; one portion was formalin fixed, paraffin embedded, and subjected to histological evaluation and immunohistochemical analysis; the other portion was fixed in 4% paraformaldehyde in PBS at 4°C overnight, embedded in paraffin, cut into 4-µm strips, and transferred to silane-coated slides (Sigma, St. Louis, MO) for ISH.

Histopathological Grading.
Formalin-fixed and paraffin-embedded liver tissue sections were subjected to histological evaluation by light microscopy including staining by H&E. Each section was reviewed by two pathologists specializing in hepatology. Histological differentiation grades for HCC were determined according to the scale of Edmondson and Steiner (22) . Grades I or I-II were defined as WELL, II or II-III as MOD, and III or III-IV as POR.

Immnohistochemistry.
Immunohistochemical analysis for formalin-fixed, paraffin-embedded tissue samples was performed using an avidin-biotin-peroxidase complex technique after microwave antigen retrieval as described previously (23) . Each section was treated with blocking solution, antihuman ANG monoclonal antibody (1:100 dilution; Santa Cruz Biotechnology, CA) or normal mouse IgG (Dako, Glostrup, Denmark), biotinylated secondary antibody, and a peroxidase-avidin complex (ABC-kit; Vector Laboratories, Burlingame, CA). The intensity of ANG immunostaining in the sections was assessed by using an AxioCam photomicroscope and the KS-400 image analyzing system (Carl Zeiss Vision GmbH, Hallbermoss, Germany) as described previously (24) . A microscopic image of each section was imported into the KS-400, in which brown-stained areas, which represented positive staining corresponding to ANG immunoreactivity, were converted into a 255-graded gray scale. The average gray scale intensity of each sample was calculated by using the KS-400 image analyzing program and was represented by the ratio to that of each sequential section immunostained by control IgG (normal mouse IgG).

MVD of HCC tissues was evaluated according to the method of El-Assal et al. (8) . with some modifications. Endothelial cells of blood vessels in HCC tissue were stained by anti-Factor VIII antibody (Dako). Positively stained endothelial cells or endothelial cell clusters that were clearly separate from adjacent microvessels and other connective tissue elements were each considered a single countable vessel and were calculated by using the KS-400.

ISH.
As a probe for human ANG cDNA, a 334-bp fragment corresponding to the mRNA coding region was generated by reverse transcription-PCR (RT-PCR) using total RNA from HepG2 cells and an oligo(dT) primer. The ANG cDNA probe was amplified by the PCR with the use of specific primers: 5'-tcctgacccagcatg-3' (nucleotides 1906–2000) and 5'-cttttaactctgcat-3' (nucleotides 2226–2240). Amplified DNA fragments were elecrophoresed on 2% agarose gel and then were purified with the Qiaquick Gel Extraction kit (Qiagen, Heiden, Germany). Then the cDNA fragment was digoxigenin-labeled with a DNA Labeling and Detection Kit (Beohringer, GmbH, Sandhofer, Germany). ISH was performed according to the manufacturer’s instructions. In addition to negative controls, the specificity of the detection was evaluated in sections that were pretreated with RNase A.

Determination of Serum ANG Concentration.
Serum ANG concentrations of HCC patients, LC patients, CH patients, and healthy subjects were determined by using an ELISA kit (Amersham), according to the manufacturer’s instructions.

Statistical Methods.
Quantitative values are expressed as means ± SD or median (range) unless otherwise indicated. Each data set was first evaluated for normality of distribution by the Komolgorov-Smirnov test to evaluate whether a nonparametric rank-based analysis or a parametric analysis should be used. Two groups were compared by either the Wilcoxon signed-rank test or by the Student t test. Multiple groups were compared by the rank-based, Kruskal-Wallis ANOVA test followed by Dunn’s test for differences among groups or a standard ANOVA followed by Dunn’s test for multiple comparisons against a control group. The correlations between variables were examined by simple or multiple linear regression analysis or Spearman’s coefficient of rank correlation test. Survival rates were calculated using the Kaplan-Meier method. Ps of <0.05 were considered to indicate statistical significance.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Characteristics of the HCC Patients.
Demographics of the 41 HCC patients studied are shown in Table 1Citation . Of these patients, 34 were positive for the HCV antibody (HCV-Ab), 4 were positive for the HbsAg and 3 were negative for both HCV-Ab and HBsAg. Thirty-four patients (83%) had LC; 18 were Child A, 20 were Child B, and 3 were Child C grade according to the Child-Pugh scoring. The median (range) tumor diameter was 23.5 mm 10–60(10–60) in WELL, 30.0 mm 10–120(10–120) in MOD and 53.5 mm 10–120(10–120) in POR. The number of patients in each stage of HCC was as follows: stage I, 4; stage II, 15; stage III, 5; stage IV, 17. For the initial therapies, HCC patients underwent hepatectomy (n = 6), TAE (n = 17), hepatic arterial infusion (HAI, n = 5) and PEI (n = 8). The criterion of successful treatment is that the normal liver tissue at the periphery of the tumor is not enhanced on computed tomography scans after 4 weeks, according to the method of Shiina et al. (25) .

ANG Expression in HCC Tissues.
Expression of the ANG protein in HCC tissues was evaluated by immunohistochemical staining followed by analysis with the KS-400 imaging system. Each pair of histologically different tissue sections was stained with anti-ANG monoclonal antibody or normal mouse IgG. As shown in Fig. 1ACitation , which represents the typical immunostaining pattern of each section, positive staining of ANG protein was observed diffusely in the cytoplasm of HCC cells as POR and MOD, but little staining was detected as a WELL-type, and almost none in the surrounding nontumorous tissue. In general, positive staining of ANG was observed in the central area or near the tumor capsule in capsulated tumors. When the intensity of the staining was assessed by computer-assisted quantitative analysis (KS-400 image analyzing system), positive incidences of the ANG staining [defined as a cutoff level >2.0 higher than the mean + 2 SD level of the control staining (stained with normal mouse IgG)] were six of six in POR cases, seven of nine in MOD cases, and two of seven in WELL cases. There was a significant correlation (P = 0.0116) between the intensity of ANG immunoreactivity and the histological differentiation grading of HCC (Fig. 2)Citation .



View larger version (89K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. Expression of ANG protein and ANG mRNA in HCC tissues. A, each tissue section was immunohistochemically stained with anti-ANG monoclonal antibody (middle panel) and normal mouse IgG (bottom panel). H&E (HE) staining is shown in top panel. B, ANG mRNA expression was examined by the ISH method as described in "Materials and Methods." A, x200; B, x400.

 


View larger version (12K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2. Correlation between tumor histological grading and the intensity of ANG immunoreactivity in HCC. Intensity of ANG immunostaining was determined by using a KS-400 image analyzing system as described in "Materials and Methods."

 
To next examine whether an increase of the ANG protein is associated with an increase of its mRNA, we performed ISH using HCC tissues (five POR, four MOD, and three WELL). The specific mRNA signals with ANG cDNA probes were homogeneously detected in the cytoplasm of HCC cells in five of five POR cases, three of four MOD cases, and one of three WELL cases. Fig. 1BCitation represents a typical example of ANG mRNA signals in a POR case (middle panel), whereas no significant hybridization signal was observed in the sections that were pretreated with RNaseA (bottom panel).

To evaluate the relationship between ANG expression and neovascularization in HCC tissues, we stained microvessels with the anti-factor VIII antibody using sequential tissue sections, and we analyzed their intensity by the KS-400 system. The number of microvessels stained heterogeneously inside the HCC tissues of MOD and POR cases clearly increased as compared with that of WELL cases (Fig. 3A)Citation . When positively stained MVD within the tumorous region was assessed by the KS-400 system, the median (range) values were 35 22–80(22–80) in WELL-type, 45 (38–92) in MOD-type, and 97.5 (60–125) in POR-type HCCs. There was a significant correlation (r = 0.877, P = 0.0009) between ANG expression levels and MVD as determined by immunohistochemical staining (Fig. 3B)Citation .



View larger version (58K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 3. Correlation between MVD and the intensity of ANG immunoreactivity in HCC. A, microvessels were immunohistochemically stained with anti-factor VIII (Anti-VIII) antibodies. x200. B, MVD was evaluated by a KS-400 image analyzing system.

 
Serum ANG Concentrations in Patients with HCC.
As shown in Fig. 4ACitation , the mean ± SD serum ANG level of LC patients (242.4 ± 126.9 ng/ml) was significantly lower than that of healthy subjects (362.3 ± 84.1 ng/ml, P = 0.0004) and that of CH patients (331.9 ± 133.8 ng/ml, P = 0.0427). HCC patients were categorized into three groups based on tumor vascularity assessed by celiac angiography (Fig. 4ACitation , insets). Mean serum ANG concentrations were 197.8 ± 64.9 ng/ml in the hypovascular group, 326.7 ± 148.6 ng/ml in the hypervascular group, and 405.0 ± 121.3 ng/ml in the very hypervascular group, the difference in the serum ANG between the three groups being statistically significant (P = 0.0108). Furthermore, a significant correlation (P = 0.0009) was observed between serum ANG concentration and tissue ANG expression level as evaluated by immunohistochemical staining (Fig. 2)Citation . Moreover, in 11 of 13 patients who underwent TAE or PEI, serum ANG concentration was significantly decreased (-21% reduction) after successful treatment of HCC (P = 0.0147), increasing once again in some recurrent cases (Fig. 4B)Citation . These findings suggested that the increased serum ANG in patients with HCC is derived from tumor cells.



View larger version (39K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 4. Serum ANG concentrations in patients with HCC, CH, and LC and in healthy subjects. A, HCC patients were divided into three groups according to celiac angiography (insets). Error bars, SE. Hypo, hypovascular; Hyper, hypervascular; Very hyper, very hypervascular; T, tumor. *, P = 0.0427; **, P = 0.0004; ***, P = 0.0108. B, serial ANG concentrations in patients with HCC during treatment with PEI and TAE. Serum ANG concentrations were decreased after successful treatments (*, P = 0.0147) and increased in four cases of recurring HCC. {circ}, PEI; •, TAE.

 
We then analyzed the relationship between serum ANG concentration and several clinicopathological parameters for HCC using univariate analyses (log-rank test). Serum ANG concentrations in patients with HCC statistically correlated with histological differential grades of HCC, tumor vascularity, and tumor size, but not with age, sex, the presence or absence of hepatitis B virus/HCV, inflammation, the presence or absence of LC, the severity of disease (the Child-Pugh grade), serum transaminase levels, platelet counts, or serum {alpha}-fetoprotein levels (Table 2)Citation .


View this table:
[in this window]
[in a new window]

  		Table 2 Relationship between serum ANG concentrations and clinicopathological features in HCC patients

 
To elucidate the implication of serum ANG levels in the prognosis of HCC, we examined the 5-year survival of each group. The Kaplan-Meier method indicated that the patients with low serum ANG levels (<=333 ng/ml, below the mean level of all HCC patients) had a significantly (P = 0.046) better 5-year survival (18 versus 0%) compared with those in the group who showed higher serum ANG levels (>333 ng/ml; Fig. 5Citation ).



View larger version (14K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 5. Overall survival rate in HCC patients. Low ANG group, patients with serum ANG levels <=333 ng/ml; High ANG group, patients with serum ANG levels >333 ng/ml.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study demonstrated increased ANG expression in MOD- and POR-type HCCs and a positive correlation between its expression levels and tumor vascularity evaluated by MVD or angiography, suggesting that ANG contributes to the neovascularization of HCC.

Previous studies have shown that large-sized (>20 mm) HCC with dedifferentiated histological types (MOD and POR) usually display the development of tumor vessels (26, 27, 28) . Authors of these studies speculated that a rapid proliferation of tumor cells in HCC eventually lead to local hypoxia, which may be a stimulus for the synthesis of angiogenic factor(s). In fact, up-regulation of VEGF gene expression via hypoxia-inducible factor-1{alpha} (HIF-1{alpha}) has been demonstrated (29) . However, a recent report revealed that VEGF expression was increased even in WELL-type HCC and decreased during dedifferentiation to MOD and POR (30) . In our study, the positive correlation between tumor size and expression level of ANG in addition to the intense expression of ANG in POR and WELL-type HCC adjacent to the tumor capsule or the central portion of tumorous tissues suggests that expression of ANG may be up-regulated in accordance with a hypoxic condition within the tumor. Accordingly, during HCC development, transcriptional regulation of ANG differs from that of VEGF, VEGF contributing to the early stages of angiogenesis and ANG to the late stages of angiogenesis.

Because ANG was detectable in sera of healthy subjects (31) , we then explored the clinical implications of serum ANG in CH, LC, and HCC. Serum ANG levels in patients with LC were significantly lower than those of CH patients, which were almost the same as in healthy subjects. In LC patients, a decrease of serum ANG levels would suggest reduced hepatic reservoirs of ANG because of a decrease of the number of hepatocytes, which are the main sources for serum ANG in normal subjects. In HCC patients, despite the cirrhotic background, serum ANG elevated as the vascularity increased from hypo to hyper and from hyper to very hyper status. Furthermore, serum ANG concentration decreased after successful treatment of HCC and increased in some cases of recurrent HCC, indicating that the measurement of serum ANG concentration may be clinically useful in monitoring the treatment efficacy of HCC, particularly in {alpha}-fetoprotein-negative cases or cases negative for protein induced by vitamin K absence or antagonist-II (PIVKA-II). The fact that the patients with the concentrations higher than 333 ng/ml had a worse prognosis supports the previous notion that the higher the vascularity, the worse the prognosis (8 , 25 , 27 , 32) .

To date, antiangiogenic therapy is one of the strategies for cancer therapy. Monoclonal antibodies against ANG have been shown to significantly suppress established human colon adenocarcinoma cells and breast carcinoma cells implanted into athymic mice (33, 34, 35, 36) . Additional in vitro and in vivo studies need to be done on whether inhibition of angiogenesis by using anti-ANG antibodies or anti-ANG receptor antibodies would be a therapeutic option for molecular targeting of HCC.


   ACKNOWLEDGMENTS
 
We thank Kevin S. Litton for editorial assistance of this manuscript, Drs. H. Iwaki and T. Ikeda for pathological review, and E. Takagawa for technical assistance in processing the image analysis.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported in part by a Research Grant for Science from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

1 To whom requests for reprints should be addressed, at Fourth Department of Internal Medicine, Sapporo Medical University School of Medicine, South-1, West-16, Chuo-ku, Sapporo, 060-8543, Japan. Phone: 81-11-611-2111; Fax: 81-11-612-7987; E-mail: jkato{at}sapmed.ac.jp Back

2 The abbreviations used are: HCC, hepatocellular carcinoma; VEGF, vascular endothelial growth factor; MVD, microvessel density; ANG, angiogenin; CH, chronic hepatitis; LC, liver cirrhosis; ISH, in situ hybridization; WELL, well-differentiated HCC; MOD, moderately differentiated HCC; POR, poorly differentiated HCC; HCV, hepatitis C virus; HbsAg, hepatitis B surface antigen; TAE, transcatheter arterial embolization; PEI, percutaneous ethanol injection. Back

Received 11/ 8/02; revised 4/21/03; accepted 6/25/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Freeny P. C., Marks W. N. Patterns of contrast enhancement of benign and malignant hepatic neoplasms during bolus dynamic and delayed CT. Radiology, 160: 613-618, 1986.[Abstract/Free Full Text]
  2. Bhattacharya S., Davidson B., Dhillon A. P. Blood supply of early hepatocellular carcinoma. Semin. Liver Dis., 15: 390-401, 1995.[Medline]
  3. Miura H., Miyazaki T., Kuroda M., Oka T., Machinami R., Kadama T., Shibuya M., Makuuchi M., Yazaki Y., Ohnishi S. Increased expression of vascular endothelial growth factor in human hepatocellular carcinoma. J. Hepatol., 27: 854-861, 1997.[CrossRef][Medline]
  4. Torimura T., Sata M., Ueno T., Kin M., Tsuji R., Suzaku K., Hashimoto O., Sugawara H., Tanikawa K. Increased expression of vascular endothelial growth factor in associated with tumor progression in hepatocellular carcinoma. Hum. Pathol., 29: 986-991, 1998.[CrossRef][Medline]
  5. Poon R. T., Ng I. O., Lau C., Zhu L. X., Yu W. C., Lo C. M., Fan S. T., Wong J. Serum vascular endothelial growth factor predicts venous invasion in hepatocellular carcinoma: a prospective study. Ann. Surg., 223: 227-235, 2001.
  6. Jin-no K., Tanimizu M., Hyodo I., Nishikawa Y., Hosokawa Y., Endo H., Doi T., Mandai K., Ishizuka H. Circulating platelet-derived endothelial cell growth factor increases in hepatocellular carcinoma patients. Cancer (Phila.), 82: 1260-1267, 1998.
  7. Poon R. T., Ng I. O., Lau C., Yu W., Fan S. T., Wong J. Correlation of serum basic fibroblast growth factor levels with clinicopathologic features and postoperative recurrence in hepatocellular carcinoma. Am. J. Surg., 182: 298-304, 2001.[CrossRef][Medline]
  8. El-Assal O. N., Yamanoi A., Soda Y., Yamaguchi M., Igarashi M., Yamamoto A., Nabika T., Ngasue Clinical significance of microvessel density and vascular endothelial growth factor expression in hepatocellular carcinoma and surrounding liver: possible involvement of vascular endothelial growth factor in the angiogenesis of cirrhotic liver. Hepatology, 27: 1554-1562, 1998.[CrossRef][Medline]
  9. Shimada M., Hasegawa H., Rikimaru T., Gion T., Hamatsu T., Yanashita Y., Shirabe K., Sugimachi K. The significance of thymidine phosphorylase activity in hepatocellular carcinoma and chronic diseased livers: a special reference to liver fibrosis and multicentric tumor occurrence. Cancer Lett., 148: 165-172, 2000.[CrossRef][Medline]
  10. Mise M., Arii S., Higashitsuji H., Furutani M., Niwano M., Harada T., Ishigami S., Toda Y., Nakayama H., Fukumoto M., Fujita J., Imamura M. Clinical significance of vascular endothelial growth factor and basic fibroblast growth factor gene expression in liver tumors. Hepatology, 23: 455-464, 1996.[CrossRef][Medline]
  11. Weiner H. L., Weiner L. H., Swain J. L. Tissue distribution and developmental expression of the messenger RNA encoding angiogenin. Science (Washington DC), 237: 280-282, 1987.[Abstract/Free Full Text]
  12. Burgmann H., Hollenstein U., Maca T., Zedwitz-Liebenstein K., Thalhammer F., Koppensteiner R., Ehringer H., Graninger W. Increased serum laminin and angiogenin concentrations in patients with peripheral arterial occlusive disease. J. Clin. Pathol., 49: 508-510, 1996.[Abstract/Free Full Text]
  13. Malamitsi-Puchener A., Sarandakou A., Dafogianni C., Tziotis J., Bartsocas C. S. Serum angiogenin levels in children and adolescents with insulin-dependent diabetes mellitus. Pediatr. Res., 43: 798-800, 1998.[Medline]
  14. Shimoyama S., Yamasaki K., Kawahara M., Kaminishi M. Increased serum angiogenin concentration in colorectal cancer is correlated with cancer progression. Clin. Cancer Res., 5: 1125-1130, 1999.[Abstract/Free Full Text]
  15. Shimoyama S., Gansauge F., Gansauge S., Negri G., Oohara T., Beger H. G. Increased angiogenin expression in pancreatic cancer is related to cancer aggressiveness. Cancer Res., 56: 2703-2706, 1996.[Abstract/Free Full Text]
  16. Miyake H., Hara I., Yamanaka K., Gohji K., Arakawa S., Kamidono S. Increased angiogenin expression in the tumor tissue and serum of urothelial carcinoma patients in related to disease progression and recurrence. Cancer (Phila.), 86: 316-324, 1999.
  17. Hartmann A., Kunz M., Köstlin S., Gillitzer R., Toksoy A., Bröcker E. B., Klein C. E. Hypoxia-induced up-regulation of angiogenin in human malignant melanoma. Cancer Res., 59: 1578-1583, 1999.[Abstract/Free Full Text]
  18. Barton D. P. J., Cai A., Wendt K., Young M., Gamero A., De Cesare S. Angiogenic protein expression in advanced epithelial ovarian cancer. Clin. Cancer Res., 3: 1579-1586, 1997.[Abstract]
  19. Montero S., Guzmán C., Cortés-Funes H., Colomer R. Angiogenin expression and prognosis in primary breast carcinoma. Clin. Cancer Res., 4: 2161-2168, 1998.[Abstract]
  20. Sobin L. H., Wittekind Ch. . TNM Classification of Malignant Tumours, UICC, ed. 5 74-77, Wiley-Liss New York 1997.
  21. Honda H., Ochiai K., Adachi E., Yasumori K., Hayashi T., Kawashima A., Fukuya T., Gibo M., Matsumata T., Tsuneyoshi M., Masuda K. Hepatocellular carcinoma: correlation of CT, angiographic, and histopathologic findings. Radiology, 189: 857-862, 1993.[Abstract/Free Full Text]
  22. Edmondson H. A., Steiner P. E. Primary carcinoma of the liver: a study of 100 cases among 48900 necropsies. Cancer (Phila.), 7: 462-503, 1954.
  23. Shi S., Key M. E., Kalra K. L. Antigen retrieval in formalin-fixed, paraffin-embedded tissues; an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J. Histochem. Cytochem., 39: 741-748, 1991.[Abstract]
  24. Kato J., Kobune M., Nakamura T., Kuroiwa G., Takada K., Takimoto R., Sato Y., Fujikawa K., Takahashi M., Takayama T., Ikeda T., Niitsu Y. Normalization of elevated hepatic 8-hydroxy-2'-deoxyguanosine levels in chronic hepatitis C patients by phlebotomy and low iron diet. Cancer Res., 61: 8697-8702, 2001.[Abstract/Free Full Text]
  25. Shina S., Tagawa K., Unuma T., Terano A. Percutaneous ethanol injection therapy for the treatment of hepatocellular carcinoma. Am. J. Roentgenol., 154: 947-951, 1989.
  26. Tanigawa N., Lu C., Mitsui T., Miura S. Quantitation of sinusoid-like vessels in hepatocellular carcinoma: its clinical and prognostic significance. Hepatology, 26: 1216-1223, 1997.[CrossRef][Medline]
  27. Park Y. N., Kim Y., Yang K. M., Park C. Increased expression of vascular endothelial growth factor and angiogenesis in the early stage of multistep hepatocarcinogenesis. Arch. Pathol. Lab. Med., 124: 1061-1065, 2000.[Medline]
  28. Morinaga S., Imada T., Shimizu A., Akaike M., Sugimasa Y., Takemiya S., Takanashi Y. Angiogenesis in hepatocellular carcinoma as evaluated by {alpha} smooth muscle actin immunohistochemistry. Hepatogastroenterology, 48: 224-228, 2001.[Medline]
  29. Forsythe J. A., Jiang B. H., Iyer N. V., Agani F., Leung S. W., Koos R. D., Semenza G. L. Activation of vascular endothelial growth factor gene expression by hypoxia-inducible factor-1. Mol. Cell. Biol., 16: 4604-4613, 1996.[Abstract]
  30. Yamaguchi R., Yano H., Iemura A., Ogasawara S., Haramaki M., Kojiro M. Expression of vascular endothelial growth factor in human hepatocellular carcinoma. Hepatology, 28: 68-77, 1998.[CrossRef][Medline]
  31. Shapiro R., Strydom D. J., Olson K. A., Vallee B. L. Isolation of angiogenin from normal human plasma. Biochemistry, 26: 5141-5146, 1987.[CrossRef][Medline]
  32. Poon R. T., Ng I. O., Lau. C., Yu W. C., Yang Z. F., Fan S. T., Wong J. Tumor microvessel density as a predictor of recurrence after resection of hepatocellular carcinoma: a prospective study. J. Clin. Oncol., 20: 1775-1785, 2002.[Abstract/Free Full Text]
  33. Olson K. A., French T. C., Vallee B. L., Fett J. W. A monoclonal antibody to human angiogenin suppresses tumor growth in athymic mice. Cancer Res., 54: 4576-4579, 1994.[Abstract/Free Full Text]
  34. Piccoli R., Olson K. A., Vallee B. L., Fett J. W. Chimetric anti-angiogenin antibody cAb 26-2F inhibits the formation of human breast cancer xenografts in athymic mice. Proc. Natl. Acad. Sci. USA, 95: 4579-4583, 1998.[Abstract/Free Full Text]
  35. Olson K. A., Fett J. W., French T. C., Key M. E., Vallee B. L. Angiogenin antagonists prevent tumor growth in vivo. Proc. Natl. Acad. Sci. USA, 92: 442-446, 1995.[Abstract/Free Full Text]
  36. Olson K. A., Beyers H. R., Key M. E., Fett J. W. Inhibition of prostate carcinoma establishment and metastatic growth in mice by an antiangiogenin monoclonal antibody. Int. J. Cancer, 98: 923-929, 2002.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Ann. Surg. Oncol.Home page
R. W. C. Pang, J. W. Joh, P. J. Johnson, M. Monden, T. M. Pawlik, and R. T. P. Poon
Biology of Hepatocellular Carcinoma
Ann. Surg. Oncol., April 1, 2008; 15(4): 962 - 971.
[Abstract] [Full Text] [PDF]

Home page
 Eur Heart JHome page
A. Tello-Montoliu, F. Marin, J. Patel, V. Roldan, L. Mainar, V. Vicente, F. Sogorb, and G. Y.H. Lip
Plasma angiogenin levels in acute coronary syndromes: implications for prognosis
Eur. Heart J., December 2, 2007; 28(24): 3006 - 3011.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
N. Yoshioka, L. Wang, K. Kishimoto, T. Tsuji, and G.-f. Hu
A therapeutic target for prostate cancer based on angiogenin-stimulated angiogenesis and cancer cell proliferation
PNAS, September 26, 2006; 103(39): 14519 - 14524.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
T. M. Katona, B. L. Neubauer, P. W. Iversen, S. Zhang, L. A. Baldridge, and L. Cheng
Elevated Expression of Angiogenin in Prostate Cancer and Its Precursors
Clin. Cancer Res., December 1, 2005; 11(23): 8358 - 8363.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hisai, H.
Right arrow Articles by Niitsu, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hisai, H.
Right arrow Articles by Niitsu, Y.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online