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Tumor Biology |
Department of Surgery, Medical Institute of Bioregulation, Kyushu University, Beppu 874-0838, Japan [T. E., H. I., M. M.]; Department of Surgery II, Kyushu University, Fukuoka, Japan [S. T.]; Division of Digestive Disease and Nutrition, University of Massachusetts Medical School, Worcester, MA [G. F. B.]; and Department of Surgery I, Faculty of Medicine, Oita Medical University, Oita, Japan [T. E., S. K.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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There have been many studies attempting to isolate the molecular mediators of tumor angiogenesis. Until recently, most of the work in the field has focused on polypeptide growth factors such as fibroblast growth factor and VEGF, which are mitogenic for ECs in vitro, and which produce an angiogenic response in vivo (7, 8, 9, 10) . Ang-1 and its naturally occurring antagonist, Ang-2, are novel ligands that regulate tyrosine phosphorylation of the Tie2/Tek receptor on ECs (11 , 12) . Proper regulation of Tie2/Tek is absolutely required for normal vascular development, seemingly by regulating vascular remodeling and EC interactions with supporting pericytes/smooth muscle cells (12, 13, 14, 15) . Recently, it has been reported that the expression pattern of Ang-2 is strongly associated with the expression of VEGF in the process of tumor angiogenesis and, subsequently, in tumor expansion (16, 17, 18, 19) .
Angs are mainly produced by ECs and pericytes, and their receptor Tie2/Tek is also expressed in ECs and partly in hematopoietic cells (13) . In particular, Ang-2 is expressed in ECs of tumor-associated vessels (18 , 20) . Therefore, Angs, especially in the Ang-2 and Tie system, may act via an autocrine manner in ECs in tumor angiogenesis. However, we have recently found that hypervascular hepatomas with aberrant vasculature show high levels of Ang-2 expression in their epithelium, and Ang-2-transfected human hepatocellular carcinoma cells may contribute to tumor development and extensive hemorrhage in nude mice (21) . With respect to gastrointestinal tumors, there have not been any studies that establish a causal role for Angs. In this study, to define a putative role for the Ang/Tie2 system in gastric cancer angiogenesis, we investigated the expression pattern of Angs and its clinical significance. On the basis of the clinical results, we examined further the biological behavior of Ang-2 stably transfected human gastric cancer cells in culture and after orthotopic implantation into nude mice. We focused especially on the role of proteases such as MMPs with regard to the possible mechanism resulting in hypervascular conditions induced by Angs in tumor angiogenesis.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Clinical Samples.
Fresh surgical specimens were obtained from 85 patients with primary gastric cancer and their paired adjacent normal gastric mucosa. They had undergone surgery at the Department of Surgery, Medical Institute of Bioregulation, Kyushu University (Beppu, Japan) from 1988 to 1997. None of these patients received preoperative treatment such as radiation and chemotherapy, although, 28 patients received postoperative adjuvant chemotherapy. Data concerning patient outcome, including overall survival and development of metastases, were available for all 85 patients, and the observation periods ranged from 6 months to 105 months (the median follow-up period was 42.1 months). Of 85 patients, 15 had died as a result of recurrence.
RT-PCR and Northern Blot Analysis.
Total RNA was extracted using the acid guanidine phenol chloroform method, then DNase treatment and the reverse transcriptase reaction was as described previously (21)
. Each full coding sequence of Ang-1 or Ang-2 was obtained by RT-PCR and confirmed, with no mutation documented using sequence analysis. As shown in Table 1
, all primers for PCR amplification, including Ang-1, Ang-2, VEGF, and Tie-2 proteases and their inhibitors, are listed. PCR amplification was performed for 25 cycles under the following conditions: (a) denaturing at 95°C for 1 min; and (b) polymerization at 72°C for 1 min. Each annealing condition for amplification of these cDNAs is included.
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-32P labeled by random priming cDNA probes for Ang-1, Ang-2, VEGF, Tie-2, proteases and their inhibitors, and c-Ets1, which were generated by RT-PCR. Filters were exposed to autoradiography for 2 h, and the mRNA levels were quantitated using a Bio-Image analyzer BAS 1000 and corrected by the levels of GAPDH as a control.
Transfection Assays and Production of Stable Cell Lines.
Endogeneous mRNA expression of both Ang-1 and Ang-2 were not detected in the human MKN-7 gastric cancer cell line. Each Ang-1 and Ang-2 cDNA were subcloned into a pcDNA3 vector (Invitrogen) and transfected into the cell line by the lipofection method (Life Technologies, Inc.) as described (21)
. Subsequently, stable transfectants were selected with 800 µg/ml of G418 treatment. Two clones of MKN-7 cells, expressing abundant Ang-2 mRNA, and 2 clones of MKN-7 cells, expressing abundant Ang-1 mRNA, were used for the subsequent experiments. A mock-transfected clone of each cell line was used for the subsequent experiments.
Invasion and Proliferation Assays.
The invasive potential of the Ang-transfected MKN-7 cells was determined using a modified two-chamber invasion assay as described (23)
. Briefly, six-well transwell plates with an 8-µm pore size were coated with gelatin. Cells on the lower side of the membrane were stained and counted. Furthermore, we performed proliferation assays using tritiated-thymidine (1 µCi; NEN, Boston, MA) as described previously (24)
. Cells (3 x 103) were seeded on 24-well plates and cultured in RPMI 1640 in the absence or presence of FBS. The medium was changed every 48 h. All experiments were performed in triplicate.
Implantation of Tumor Tissues into Subcutis and Gastric Wall of Mice.
To investigate the tumorigenicity and biological effect of Ang-2 expression in MKN-7 cells, we injected Ang-2 transfectant cells (1 x 104) in 50 µl of PBS into the subcutis of nude mice. Tumors in the exponential phase were resected and necrotic tissue was removed. The viable tumor tissue was cut into pieces 2 mm in diameter. Under anesthesia with diethylether, the tumor piece was fixed on the anterior wall of the stomach by a superficial serosal suture with 6-0 propylene (Ethicon, Somerville, NJ). Parent MKN-7 tumors and control vector-transfected MKN-1 tumors were used as controls. Nude mice (n = 12) were analyzed for histological examination or detection of mRNA of tumor at 4 weeks after implantation. The macroscopic number and size of metastases to lung, liver, mesentery, other abdominal organs, and lymph nodes were evaluated.
In Situ Hybridization.
Paraffin sections from 30 samples of human gastric cancer were studied. Antisense RNA probes were generated using SP6 RNA polymerase and labeled with immunofluorescence using an RNA color kit (Amersham International) as described (25)
. Sense probes were used as the negative control.
Immunoblot Analysis and Immunohistochemistry.
To evaluate the protein, Western blot analysis was performed using a polyclonal antibody against Ang-1 or Ang-2 (Santa Cruz Biochemicals) as described previously (24)
. Next, to determine the localization of Ang-1, Ang-2, Tie-2, and proteases including MMP-1, MMP-9, and uPA in cancer tissue specimens, an immunohistochemical analysis was performed as described previously (24)
. ECs were detected by vWF antibody (DAKO, Kyoto, Japan), and vascular smooth muscle cells or pericytes were detected by -SMA antibody (Sigma). Antibodies against MMP-1, MMP-9, and uPA were purchased from Fuji Chemical Industries (Takaoka, Japan). These proteins were detected using the avidin-biotin-peroxidase method (LSAB Kit; DAKO, Kyoto, Japan). Furthermore, these enzymes were also detected using immunofluorescence microscopy.
Microvessel Count and Vessel Maturation Index.
The microvessel count was quantitatively examined as described previously (26)
. The fraction of blood vessels found to be associated with both -SMA-positive periendothelial cells and vWF-positive ECs was defined as the vessel maturation. This measure was determined by scoring blood vessels larger than capillaries (i.e., vessels containing a lumen large enough for several erythrocytes), because capillaries are sparsely coated by pericytes and, therefore, may falsely appear as uncovered in these sections. Quantification of the type of vessel in several high-power fields of each tumor specimen allowed the assignment of a vessel maturation index to each tumor.
Coculture Assay of HUVECs and Ang-1- or Ang-2-Transfectant MKN-7 Cells with or without Exogenous VEGF.
In this study, 1 x 103 or 1 x 105 HUVECs were cultured in two-chamber, six-well plates (Falcon Laboratories, McLean, VA). After HUVECs attached to the lower chamber, the medium was changed to RPMI 1640 without FBS used as a CM. Ang-1- or Ang-2-transfectant MKN-7 cells (1 x 105) were incubated in the upper chamber with an 8-µm pore size for 12, 24, and 48 h. Furthermore, these conditions were divided into two subgroups: with or without recombinant human VEGF 165 (R&D). Concentrations of recombinant VEGF added into the supernatant of cocultured medium ranged from 10 to 100 ng/ml. After coculture with Ang-1- or Ang-2-transfectant, HUVECs were harvested and used for additional examination.
Statistical Analysis.
The BMDP Statistical Package program (BMDP, Los Angeles, CA) for the main frame computer (4381; IBM, Armonk, NY) was used for all analyses. Associations between the variables were tested by Student’s t test or by Fisher’s exact probability test. The BMDP PIL program was used for survival analysis (Kaplan-Meier method) and for testing the equality of the survival curves (Mantel-Cox method). The BMDP P2L program was used for multivariate adjustments for some covariates, simultaneously, with Cox’s proportional hazards model. Model selection was performed using the forward stepwise method. All statistical differences were deemed significant at the level of P < 0.05.
The histopathological type and staging of gastric carcinomas were classified on the basis of the criteria set up by the Japanese Society for Cancer of the Stomach (27) .
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Fig. 1
shows representative cases. In particular, the expression of Ang-2 mRNA in 75% of the 85 cases was greater in tumor tissue (T) than normal tissue (N). The average levels of Ang-2 mRNA expression in groups showing increased vascular involvement and advanced stage were significantly higher than those of the other groups (Table 3)
. However, there was no significant correlation between each level of Ang-1 mRNA, Tie-2 mRNA, and clinicopathological features (data not shown).
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. Using these averages, we classified expression into two groups: a high group (n = 43) and a low group (n = 42). With regard to prognosis, a difference in survival time was significant between the high Ang-2 mRNA-expression group and the low Ang-2 mRNA-expression group (P < 0.05; Mantel-Cox method; Fig. 2
). On the other hand, there was no significant correlation between the levels of Ang-1 mRNA or Tie-2 mRNA expression and survival time (data not shown). In a multivariate analysis for prognosis, parameters included vascular involvement, lymph node metastasis, clinical stage, postoperative adjuvant chemotherapy, and Ang-2 mRNA expression. This analysis demonstrated that high Ang-2 mRNA expression was not an independent prognostic factor (Hazard ratio, 1.54; 95%CI, 1.01–2.15).
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. Ang-2 was detected in cancer cells of both intestinal and diffuse types of gastric cancer not forming a solid mass. Using in situ hybridization, transcripts of Ang-2 were found in cancer epithelial cells (Fig. 3, F and G)
. In addition, there was a significant association between Ang-2 mRNA and VEGF mRNA expressions in cancer tissues by Northern blot analysis using Student’s t test (data not shown).
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, the mRNA levels of six gastric cancer cell lines and the EC line used in this study are listed. We used the MKN-7 cell line for additional experiments. First, we transfected Ang-2 expression vectors into MKN-7 gastric cancer cells and selected stable clones. Ang-2 mRNA expression and protein expression were confirmed by Northern and Western blot analyses, respectively. Western blot analysis showed that Ang-2 protein was found in cell lysates of transfected MKN-7 cells and in the supernatant of cultured medium (Fig. 4A
, right). With regard to Ang-1, transfection assays and the evaluation of Ang-1 protein were performed similarly (Fig. 4A
, left).
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. These findings thus demonstrated that the addition of exogenous Ang-2 did not alter cell proliferation of the gastric cancer cell lines. Furthermore, invasion assays revealed that addition of exogenous Ang-2 did not alter cell invasiveness of the gastric cancer cell lines (data not shown). After transfected cells were injected into the subcutis of nude mice, tumors arising from Ang-2-transfected MKN-7 cells developed large and marked hemorrhages compared with tumors from mock-transfected clones (Fig. 4C
, a and c). There was a significant association between Ang-2 expression and macroscopic tumor growth in vivo (Table 5)
. To investigate the organ-microenvironment influence on tumor growth and metastasis, tumors were then transplanted into an orthotopic site (gastric wall) of nude mice. Ang-2-transfected cells formed tumors in 10 of 12 nude mice, whereas mock-transfected clones formed tumors in 2 of 12 nude mice. We also found peritoneal dissemination and liver metastasis of tumors only in the Ang-2-transfectant group (Fig. 4C
, d and e). Immunohistochemical examination of the tumors derived from Ang-2 transfectants revealed that a huge mass was formed in the subserosal space of the gastric wall (Fig. 4D
, a and b). In addition, Ang-2 was strongly expressed in cancer cells and also partly expressed in ECs at sites surrounding the cancer cells (Fig. 4D
, c). On the other hand, Ang-2 was not detected in cancer cells of the mock-transfectant tumors (Fig. 4D
, d). Its receptor, Tie-2, was only expressed in ECs at sites surrounding the cancer cells (Fig. 4D
, e).
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. Next, to evaluate the relationship between expression of Ang-2 and vessel maturation in these tumors, we determined the vessel maturation index, defined as the fraction of vessels that are associated with -SMA-positive cells around ECs (Fig. 5, A and B)
. The maturation index in the primary and metastatic liver tumors resulting from the Ang-2 transfectants was significantly lower than that of parent and/or control vector-transfected cell tumors (Fig. 5C)
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. VEGF protein was also detected in culture medium and subsequently in CM with added recombinant VEGF (10, 50, and 100 ng/ml) from parental MKN-7 cells (Fig. 6A)
. Fifty ng/ml of recombinant VEGF was added into the coculture medium.
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. Next, we examined the cellular expression and distribution of MMP-1, MMP-9, and uPA in ECs by immunofluorescence microscopy. Similar to the mRNA expression of these enzymes, they were strongly induced only by Ang-2 in the presence of VEGF (Fig. 6D)
. Finally, mRNA expression of c-Ets1, which is a promoter of MMP-1, MMP-9, or uPA, was examined by Northern blot analysis (Fig. 7A)
. The mRNA expression of c-Ets1 in ECs was up-regulated by Ang-2 in the presence of VEGF (Fig. 7B)
. In addition, different cell numbers of HUVECs did not influence the fold induction of the proteases and c-Ets1 mRNA expression.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recently, it has been reported that many tumors rapidly co-opt existing host vessels to form an initially well-vascularized tumor mass, and Ang-2 plays an important role in the process (17 , 31) . In tumors forming a solid mass, such as glioblastoma or astrocytoma, Ang-1 is expressed in tumor cells and Ang-2 is strikingly induced in co-opted vessels adjacent to the tumor cells (16 , 18) . It is considered that the intense autocrine expression of Ang-2 by ECs in tumor-associated vessels contributes to tumor expansion. In this study, we investigated the patterns of Ang-2 expression in clinical samples of gastric carcinoma by immunohistochemistry. We determined that Ang-2 was expressed not only in ECs in tumor-associated vessels, but also in cancer cells in both the intestinal type forming a solid mass and in the diffuse type of gastric cancer not forming a solid mass. This result suggests that Ang-2 derived from cancer cells may undergo the regression of tumor-associated vessels and robust angiogenesis.
To assess the biological role of Ang-2 in the tumor angiogenesis of gastric cancer, we performed transfection experiments and analyzed the tumorigenicity and metastatic potential of Ang-2 transfected into MKN-7 cells in nude mice. As a result, Ang-2-expressing MKN-7 cells developed tumors with a highly metastatic potential with hypervascularity. The initiation of new-vessel formation needs destabilization of the mature structure of vessels and, subsequently, degradation of the extracellular matrix surrounding ECs. Vessels that are formed in the marked hypervascular tumors producing Ang-2 tend to be immature. In fact, the immaturity of the vessels and the microvascular counts in these tumors were significantly associated with Ang-2 production. However, it has not been clarified how the process from destabilization of vessels to degradation of the extracellular matrix is regulated. In this study, we focused on proteinases that are required to degrade the extracellular matrix (32, 33, 34, 35, 36) . We investigated whether the expression of proteases of ECs in tumor angiogenesis was regulated by Ang-2. We also evaluated the influence of VEGF on the role of Ang-2, because there was a significant correlation between mRNA expression levels of Ang-2 and VEGF in tumor tissues of human gastric cancer. In ECs constitutively expressing the Tie-2 receptor, mRNA and the protein expression of MMP-1, MMP-9, and uPA were significantly up-regulated by Ang-2 in the presence of VEGF; in contrast, these were not significantly up-regulated by Ang-1 or Ang-2 alone. On the other hand, the expression of these enzymes was not significantly altered in cell lines lacking Tie-2 expression, such as the Ang-1 or Ang-2 transfectants. The different protease expression pattern between ECs and Tie-2-nonexpressing cells is ascribed to the Ang/Tie-2 pathway. These properties suggest that induction of proteases in ECs by Ang-2 together with VEGF during tumor angiogenesis could be partly regulated not only in an autocrine manner but also in a paracrine manner via the Ang/Tie-2 pathway.
Why does Ang-2 require the presence of VEGF to induce these proteases compared with Ang-2 or VEGF or Ang-1 alone? Because Ang-2, unlike Ang-1, cannot induce autophosphorylation of the Tie-2 receptor in human ECs, Ang-2 is considered to inhibit Ang-1/Tie-2 activity. Induction of MMP-1, MMP-9, and uPA is regulated by common promoter regions such as c-Ets1 (37 , 38) . It has recently been reported that Tie promoter activity is also controlled by EC Ets factors (39) . We therefore consider that Ets-1 may play a key role in tumor angiogenesis via the Ang/Tie system. We hypothesized that the signaling by Ang-1 via the Tie-2 receptor may act as a negative regulator of c-Ets1 activity and result in the suppression of the production of these proteases; once Ang-2 blocks the activation of Tie-2 by Ang-1, subsequent c-Ets1 activity may be increased. In fact, c-Ets1 mRNA expression in ECs was significantly up-regulated by Ang-2 in the presence of VEGF by Northern blot analysis. Interestingly, up-regulation of MMP-1 and MMP-9, uPA, or c-Ets1 mRNA expression by Ang-2 was inhibited by Ang-1. This result suggests that Ang-1 competes with Ang-2 for up-regulation of these enzymes via c-Ets1. The role of VEGF in tumor angiogenesis is to activate c-Ets1 and, in addition, to promote the proliferation and cell growth of ECs (37 , 40 , 41) . On the other hand, Ang-2 could induce metalloproteinases to degrade vessel basement membranes via activation of c-Ets1. Furthermore, this induction might be enhanced in the presence of VEGF. The implication is that VEGF acts as a powerful promoter of tumor angiogenesis and results in tumor progression, and Ang-2 acts as an initiator to begin neovascular formation. In malignant diseases such as gastric carcinoma, the dynamic balance between Ang-2 and VEGF might be impaired. Thus, overexpressed Ang-2 together with VEGF derived mainly from cancer cells might promote tumor angiogenesis in gastric cancer development. Recent studies have demonstrated that Ang-2 mRNA levels were increased by VEGF or hypoxia in bovine microvascular ECs (42) , although the detailed regulation of Ang-2 mRNA levels in gastric cancer cells remains unclear and will require additional investigation for clarification.
In conclusion, our findings demonstrate that Ang-2 is produced not only in ECs, but also in cancer cells in human gastric carcinoma. On the basis of the experiments in this study, Ang-2 produced from gastric cancer cells may play an important role in tumor angiogenesis in the presence of VEGF.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This study was supported in part by the Ministry of Education, Culture, and Science of Japan (Grants 11139252, 10470260, and 10671198). 
2 To whom requests for reprints should be addressed, at Department of Surgery, Medical Institute of Bioregulation, Kyushu University, 4546 Tsurumihara, Beppu 874-0838, Japan. Phone: 81-977-27-1650; Fax: 81-977-27-1651; E-mail: mmori{at}tsurumi.beppu.kyushu-u.ac.jp
3 EC, endothelial cell; VEGF, vascular endothelial growth factor; Ang, angiopoietin; MMP, matrix metalloproteinase; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HUVEC, human umbilical venous endothelial cell; RT-PCR, reverse transcription-PCR; vWF, von Willebrand factor; uPA, urokinase-type plasminogen activator; SMA, smooth muscle actin; PAI, plasminogen activator inhibitor; CM, conditioned medium.
Received 6/12/00. Accepted 1/ 2/01.
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 N. Giuliani, S. Colla, M. Lazzaretti, R. Sala, G. Roti, C. Mancini, S. Bonomini, P. Lunghi, M. Hojden, G. Genestreti, et al. Proangiogenic properties of human myeloma cells: production of angiopoietin-1 and its potential relationship to myeloma-induced angiogenesis Blood, July 15, 2003; 102(2): 638 - 645. [Abstract] [Full Text] [PDF]
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L. Zhang, N. Yang, J.-W. Park, D. Katsaros, S. Fracchioli, G. Cao, A. O'Brien-Jenkins, T. C. Randall, S. C. Rubin, and G. Coukos Tumor-derived Vascular Endothelial Growth Factor Up-Regulates Angiopoietin-2 in Host Endothelium and Destabilizes Host Vasculature, Supporting Angiogenesis in Ovarian Cancer Cancer Res., June 15, 2003; 63(12): 3403 - 3412. [Abstract] [Full Text] [PDF]
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R. R. White, S. Shan, C. P. Rusconi, G. Shetty, M. W. Dewhirst, C. D. Kontos, and B. A. Sullenger Inhibition of rat corneal angiogenesis by a nuclease-resistant RNA aptamer specific for angiopoietin-2 PNAS, April 29, 2003; 100(9): 5028 - 5033. [Abstract] [Full Text] [PDF]
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T. Hashimoto, G. Wen, M. T. Lawton, N. J. Boudreau, A. W. Bollen, G.-Y. Yang, N. M. Barbaro, R. T. Higashida, C. F. Dowd, V. V. Halbach, et al. Abnormal Expression of Matrix Metalloproteinases and Tissue Inhibitors of Metalloproteinases in Brain Arteriovenous Malformations * Growth and Bleeding in BAVM: Another Role for MMPs Stroke, April 1, 2003; 34(4): 925 - 931. [Abstract] [Full Text] [PDF]
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 T. Etoh, Y. Himeno, T. Matsumoto, M. Aramaki, K. Kawano, A. Nishizono, and S. Kitano Oncolytic Viral Therapy for Human Pancreatic Cancer Cells by Reovirus Clin. Cancer Res., March 1, 2003; 9(3): 1218 - 1223. [Abstract] [Full Text] [PDF]
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F. Tanaka, S. Ishikawa, K. Yanagihara, R. Miyahara, Y. Kawano, M. Li, Y. Otake, and H. Wada Expression of Angiopoietins and Its Clinical Significance in Non-Small Cell Lung Cancer Cancer Res., December 1, 2002; 62(23): 7124 - 7129. [Abstract] [Full Text] [PDF]
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W. W. Spurbeck, C. Y. C. Ng, T. S. Strom, E. F. Vanin, and A. M. Davidoff Enforced expression of tissue inhibitor of matrix metalloproteinase-3 affects functional capillary morphogenesis and inhibits tumor growth in a murine tumor model Blood, October 16, 2002; 100(9): 3361 - 3368. [Abstract] [Full Text] [PDF]
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I. B. Lobov, P. C. Brooks, and R. A. Lang From the Cover: Angiopoietin-2 displays VEGF-dependent modulation of capillary structure and endothelial cell survival in vivo PNAS, August 20, 2002; 99(17): 11205 - 11210. [Abstract] [Full Text] [PDF]
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K. Tanaka, H. Sonoo, J. Kurebayashi, T. Nomura, S. Ohkubo, Y. Yamamoto, and S. Yamamoto Inhibition of Infiltration and Angiogenesis by Thrombospondin-1 in Papillary Thyroid Carcinoma Clin. Cancer Res., May 1, 2002; 8(5): 1125 - 1131. [Abstract] [Full Text] [PDF]
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T. Hawighorst, M. Skobe, M. Streit, Y.-K. Hong, P. Velasco, L. F. Brown, L. Riccardi, B. Lange-Asschenfeldt, and M. Detmar Activation of the Tie2 Receptor by Angiopoietin-1 Enhances Tumor Vessel Maturation and Impairs Squamous Cell Carcinoma Growth Am. J. Pathol., April 1, 2002; 160(4): 1381 - 1392. [Abstract] [Full Text] [PDF]
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E. I. Deryugina, L. Soroceanu, and A. Y. Strongin Up-Regulation of Vascular Endothelial Growth Factor by Membrane-type 1 Matrix Metalloproteinase Stimulates Human Glioma Xenograft Growth and Angiogenesis Cancer Res., January 1, 2002; 62(2): 580 - 588. [Abstract] [Full Text] [PDF]
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 Y. Mochizuki, T. Nakamura, H. Kanetake, and S. Kanda Angiopoietin 2 stimulates migration and tube-like structure formation of murine brain capillary endothelial cells through c-Fes and c-Fyn J. Cell Sci., January 1, 2002; 115(1): 175 - 183. [Abstract] [Full Text] [PDF]
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