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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online October 18, 2002 as doi:10.1096/fj.02-0509fje. |
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2
* Angiogenesis Laboratory, Department of Internal Medicine, Research Institute for Growth and Development (GROW),
Department of Radiology,
Department of Obstetrics and Gynecology,
Department of Pathology, Maastricht University and University Hospital Maastricht, Maastricht, The Netherlands; and
Department of Biochemistry, University of Minnesota Health Sciences Center, Minneapolis, Minnesota, USA
2Correspondence: Angiogenesis Laboratory, Department of Pathology, University Hospital Maastricht, P. Debyelaan 25, 6202 AZ Maastricht, The Netherlands. E-mail: a.griffioen{at}intmed.unimaas.nl
SPECIFIC AIMS
The first aim of the present study was to assess whether the novel and unique approach of the de novo design of ß-sheet scaffold cytokine-like peptides can be used to develop angiogenesis inhibitors that inhibit tumor growth in vivo. A second aim was to unravel the mechanism by which anginex exerts its angiostatic effect.
PRINCIPAL FINDINGS
1. Anginex inhibits proliferation of and induces apoptosis in mouse endothelial cells (EC)
We described the growth inhibitory effect of anginex on human EC. It has been published that this effect was EC specific (growth of normal fibroblasts and tumor cell lines was unaffected) and non-species specific. Here, the influence of anginex on proliferation of EC was analyzed using the mouse EC lines SVEC4–10 and TME and human umbilical vein EC (HUVEC). After 3 days of incubation with 75 µM anginex, proliferation was inhibited by 50% and 74% for these two cell lines, respectively.
Apoptosis induction by anginex in TME and SVEC4–10 was observed to be concentration dependent, with maximal apoptosis induction in both cell lines occurring at 75 µM. When primary cultures of mouse EC isolated from lungs and heart of mice and cultured for 3 days on a fibronectin coat with or without bFGF and in the presence or absence of 75 µM anginex, similar results were observed. In HUVEC lysates treated for 72 h with ßpep-25, significant caspase-3 activity was seen. An inhibitor of caspases (z-VAD.FMK) was able to completely inhibit anginex-induced apoptosis (Fig. 1
D). Moreover, z-VAD.FMK did not prevent the detachment of EC, suggesting that the induction of apoptosis by anginex as the result of cell detachment.
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2. Anginex inhibits the migration and tube formation of EC
Addition of anginex to wounded confluent monolayers of SVEC4–10 and TME caused migration of these cells to be inhibited in a dose-dependent manner. At a concentration of 75 µM anginex, 91% and 88% inhibition was observed 24 h after wounding the monolayer for TME cells and SVEC4–10 cells, respectively. A higher sensitivity of mouse EC relative to HUVEC at the level of EC migration was observed. Tube formation was studied using the mouse aortic ring assay. An 
70% inhibition of sprout formation in this ex vivo model was noted for anginex at 75 µM.
3. Anginex inhibits angiogenesis in vivo
In the chorioallantoic membrane (CAM) assay, which is a model for developmental angiogenesis, topically added anginex inhibited the formation of new blood vessels in a concentration-dependent way. The maximal response of 50% angiogenesis inhibition was reached at 50 µM anginex. Responses were already visible at 0.25 µM concentrations, whereas treatment with the negative control peptide ßpep-28 did not show a significant reduction in vessel density (Fig. 1A
). Tumor angiogenesis in this model, mimicked by transplantation of B16F10 mouse melanoma onto the CAM at day 10 (Fig. 1B
), was inhibited for 33% by daily treatment with anginex (P<0.00002) as measured on day 17 (Fig. 1C, D
).
4. Anginex inhibits tumor growth and angiogenesis in a mouse tumor model
A profound dose-dependent inhibition of tumor growth by anginex was observed in the B16F10 melanoma model in C57BL/6 mice. At the optimal dose of 6 mg/(kg·day) administered by continuous subcutaneous (s.c.) infusion using osmotic minipumps, tumor growth was significantly inhibited by 63% vs. that in control mice treated with saline alone. A maximal inhibition of 73% was observed on day 15. TNP-470 served as a positive control (Fig. 2
A). Anginex treatment showed no toxicity in these animals as assessed by macroscopic and behavioral determinants and body weight, as well as by histological evaluation of the organs and measurement of hematocrit (not shown).
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The tumor inhibitory effect by anginex was the result of angiogenesis inhibition as assessed by vessel density measurement, which was inhibited in the tumor for 50% relative to control tumors. A similar reduction in vessel density was found in tumors of TNP-470-treated mice (Fig. 2B-D
).
The use of magnetic resonance imaging (MRI, Fig. 2E, F
) indicated a smaller increase in signal intensity induced by the contrast agent in tumors of anginex-treated animals compared with signal intensity in tumors of control animals.
5. Anginex inhibits tumor growth and angiogenesis in a human xenograft tumor model
Treatment of mice with established (50 mm3) MA148 human ovarium carcinoma tumors by s.c. placed osmotic minipumps as of day 17 revealed a significant (n=11, P<0.004) 75–80% inhibition of tumor growth.
6. Anginex targets tumor EC
Injection of Oregon green-labeled anginex i.v. in B16F10 tumor-bearing mice and measuring the serum of these mice at different time points revealed a serum half-life of 50 min. Oregon green-labeled anginex clearly identified tumor blood vessels. Immunohistochemical detection of blood vessels using a PE-labeled anti-CD31 antibody revealed an efficient binding of anginex in tumor vessels but not in vessels of the hind-limb muscle or the kidney.
7. Anginex targets angiogenically activated EC
To find in vitro evidence for the preference of anginex to affect activated EC, anginex binding was studied on resting HUVEC (directly fixed after isolation from the umbilical cord), HUVEC activated by culture conditions, and HUVEC activated by an angiogenic factor. A clear augmented binding, demonstrated by flow cytometry using the 2D10 anti-anginex antibody, was observed with increased EC activation. To obtain functional evidence, EC were brought to a quiescent state by a thymidine block, resulting in a clear reduction of apoptosis induction by anginex.
CONCLUSIONS AND SIGNIFICANCE
The current paper demonstrates the first in vivo evidence that anginex inhibits tumor growth. It has been shown that anginex inhibits angiogenesis and specifically targets angiogenically activated tumor EC. The present results indicate that the novel approach of the de novo design of cytokine-like peptides using an 
-chemokine ß-sheet scaffold led to the identification of a therapeutically useful anti-cancer compound.
The in vitro studies suggest that angiogenesis inhibition by anginex is mediated by inhibition of adhesion and migration in activated EC, leading to induction of apoptosis. We favor this view rather than direct induction of apoptosis and subsequent detachment of EC from the matrix, because the caspase inhibitor z-VAD.FMK completely blocked apoptosis but did not prevent the detachment and anoikis of the EC. Further evidence for this view was provided by the fact that the effect of anginex on migration was seen at concentrations that were not inhibitory for proliferation of EC. These observations suggested that modulation of adhesion and/or migration of EC is the initial event ultimately resulting in modulation of proliferation and induction of apoptosis.
In the first in vivo model, the chick embryo CAM assay, anginex inhibited angiogenesis in a dose-dependent manner. After xenogenic transplantation of mouse tumor tissue onto the CAM, anginex inhibited tumor-induced angiogenesis to the level of vessel density in CAMs without a tumor.
In the syngenic murine B16F10 melanoma model in C57BL/6 mice, anginex demonstrated a profound anti-tumor effect. In the slow-growing MA148 human ovarium carcinoma model, an up to 80% inhibition of tumor growth was observed. In both tumor models, the anti-tumor activity of anginex is most likely mediated by inhibition of angiogenesis because microvessel density values in the treated tumors were significantly decreased. This was supported by MRI, where reduced signal intensity in tumors of anginex-treated animals was observed relative to tumors in control animals.
Several lines of evidence are presented for the preferential activity of anginex on activated EC. First, whereas bFGF acts as a survival factor for EC, anginex treatment in the presence of bFGF forced equivalent numbers of EC into apoptosis as treatment with anginex in the absence of bFGF. Second, anginex binds more efficiently the more an EC culture is activated. Third, to show loss of activity in quiescent EC, arresting the cells in early S-phase of cell cycle resulted in loss of 50% of apoptosis induction by anginex. Fourth, fluorescently labeled anginex was found to specifically target tumor EC but not EC in normal tissues. Therefore, anginex may be used to cotarget isotopes, toxins, or drugs. At the same time, the specific homing to angiogenically activated blood vessels may be an important tool for diagnostic use in the clinic to determine the angiogenic potential of tumors.
The current results indicate that anginex is a powerful anti-tumor angiogenesis inhibitor that shows promise of being developed to treat human cancers. Even though this paper reports only on the anti-tumor activity of anginex, the possible broader use of the peptide in treating other diseases like rheumatoid arthritis, endometriosis, atherosclerosis, psoriasis, and ocular neovascularization requires further investigation and will be the subject of future research in our laboratories.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0509fje; to cite this article, use FASEB J. (October 18, 2002) 10.1096/fj.02-0509fje 
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), 1.5 (
), 6 (
) or 12 (
) mg/(kg·day) doses of anginex, with 6 mg/(kg·day) BSA (
) or ßpep-28 (
) delivered by osmotic minipumps, or with 2 mg TNP-470 intraperitoneal (i.p.) every 2 days as of day 9 (open asterisks). Statistical significant inhibition of tumor growth (ANOVA test) was observed for anginex doses of 6 (P<0.0001) and 12 mg/(kg·day) (P<0.003) and with TNP-470 (P<0.0001). Cryosections of tumors from control (B) and 6 mg/(kg·day) anginex (C) -treated mice were stained with CD31 antibody for microvessel density (MVD) assessment. D) Quantification of MVD as mean number of vessels per 100 mm2 (±SE, *P<0.05). E, F) T2-weighted images illustrate equal-sized tumors (indicated as t) of a BSA-treated control animal (E) and an anginex-treated (6 mg·kg-1·day-1) animal (F). Subtractions of pre- and postcontrast T1-weighted tumor measurements of anginex- and BSA-treated animals are shown in the inserts. g = gut, l = left hind leg, v = ventral side of the mouse.