doi: 10.1161/01.ATV.0000068685.72914.7E
© 2003 American Heart Association, Inc.
Letters to the Editor |
Rapid Analysis of Angiogenesis Drugs in a Live Fluorescent Zebrafish Assay
Zygogen, LLC (L.M.C., M.A.C., S.L., A.L.R.), Atlanta, GA; and Department of Molecular, Cellular, and Developmental Biology (S.L., J.-N.C.), University of California, Los Angeles
To the Editor:
Solid tumors require an adequate supply of blood vessels to survive, grow, and metastasize.1–3 New blood vessels that nourish growing tumors form by angiogenesis. Drugs shown to have anti-angiogenic activity are currently in clinical cancer trials.4 To date, anti-angiogenic drugs have had mixed success in clinical application. Many new compounds may need to be tested to identify drugs capable of treating a wide range of tumors. The ideal assay for screening new compounds should involve blood vessels growing in their natural environment, such as a whole living organism, yet be amenable to rapid analysis. No current assays provide such a unique combination. We describe here an assay using the zebrafish (Danio rerio) that provides the relevance of an in vivo environment as well as the potential for high throughput drug screening.
The zebrafish has become a well accepted model for studies of vertebrate developmental biology. The vascular system has been well described and shown to be highly conserved in the zebrafish.5,6 Many zebrafish blood vessels form by angiogenic sprouting and appear to require the same proteins that are necessary for blood vessel growth in mammals. In addition, anti-angiogenic compounds, such as PTK787/ZK222584 and SU5416, have been shown to affect the formation of zebrafish blood vessels.7,8
Current methods of visualizing blood vessels in the zebrafish include whole mount in situ hybridization,9,10 detection of endogenous alkaline phosphatase activity,8 and microangiography.11 The first two methods are time consuming and involve fixation of embryos and larvae prior to analysis. Microangiography is also labor intensive and only useful for visualization of patent blood vessels in a complete circulatory system. Transgenic zebrafish with fluorescent blood vessels12,13 represent a less labor-intensive way of visualizing blood vessels in the zebrafish. We have generated a transgenic line with fluorescent blood vessels by driving expression of a green reef coral fluorescent protein (G-RCFP14) with a promoter for the vascular endothelial growth factor receptor 2 gene (VEGFR2, also referred to as Flk-1 or KDR).
VEGFR2 is one of several receptors for VEGF family members in humans and is expressed specifically in blood vessels.15 Several zebrafish VEGFR2 cDNAs have been cloned and their expression pattern described.7,9,10,16 We used a 6.5-KB genomic fragment 5' to the VEGFR2 initiation codon to drive G-RCFP expression specifically in zebrafish blood vessels. Hereafter, the stable transgenic line generated is referred to as TG(VEGFR2:G-RCFP), using standard zebrafish nomenclature. The expression pattern of G-RCFP in TG(VEGFR2:G-RCFP) embryos mirrored that of VEGFR2 in situ hybridization (not shown). Please see Figure I, available at http://atvb.ahajournals.org, for detailed fluorescent expression in TG(VEGFR2:G-RCFP) embryos.
The bright, consistent fluorescence of angiogenic blood vessels in TG(VEGFR2:G-RCFP) zebrafish embryos suggested that they could provide an ideal tool for testing angiogenesis drugs. Jain et al17 described several factors that are important for the design of an optimal angiogenesis assay, including ease of experimentation, cost-effectiveness, rapidity, reproducibility, and ability to quantify vessel formation. Pairs of adult zebrafish produce thousands of eggs routinely and are relatively inexpensive to maintain, thus fulfilling the first two criteria.
To test the utility of the TG(VEGFR2:G-RCFP) line for angiogenesis drug screening, we subjected embryos to compounds known to have anti-angiogenic activity, including SU541618,19 and SU6668,20 two indolinone-based small molecules demonstrated to inhibit VEGF-induced vascular endothelial cell proliferation in vitro. Furthermore, SU5416 and SU6668 have been reported to inhibit the formation of metastases and microvessel formation and increase apoptosis of both tumor cells and tumor endothelial cells in mouse xenograft models.21 SU5416 is believed to specifically target VEGF receptors,18,22 while SU6668 inhibits the basic fibroblast growth factor receptor and the platelet-derived growth factor receptor in addition to the VEGF receptors.20
Zebrafish embryos from the TG(VEGFR2:G-RCFP) line were incubated overnight with SU5416 and SU6668, beginning when embryos were at the 13-somite stage (approximately 15 hours post fertilization [hpf]), before angiogenic sprouting of intersegmental and head vessels had begun. As shown in parts A through C of the Figure, both compounds completely blocked intersegmental vessel formation, while preserving fluorescence in the dorsal aorta, caudal artery, caudal veins, and major cranial vessels. In addition, blood vessels in the head that form by angiogenesis were not observed in treated embryos. Treated embryos also exhibited an enlarged pericardial cavity and evidence of blood pooling in the ventral tail. These findings are reminiscent of the phenotype observed following knockdown of VEGF-A in the zebrafish by antisense morpholino,23 suggesting that these effects result from lack of a functioning vascular system rather than nonspecific toxicity. The concentrations of the compounds required to see these effects (10 µmol/L for SU5416 and 15 µmol/L for SU6668) are similar to the plasma level of SU5416 (5 µmol/L) in patients treated in clinical trials.4,19 For each compound, 136 embryos were treated in several separate experiments, and all exhibited the phenotype shown in the Figure.
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To determine whether angiogenesis could resume after the embryos are removed from the compounds, embryos were allowed to recover in fresh water for 24 to 72 hours after application of compounds. We found that blood vessels do begin to sprout again from the dorsal aorta, but they often appear irregular in shape and position, when compared with untreated embryos, as shown in part D of the Figure. Intersegmental blood vessel patterning is dependent on signals originating from the somites.24 While the somites in drug-treated embryos appeared normal, it is possible that molecules required for correct patterning of intersegmental vessels are not present at this later stage of development. Alternatively, drug treatment may affect expression of repulsive signals in the somites. Please see Figure II, available at http://atvb.ahajournals.org, for further experiments performed with these compounds.
In conclusion, the TG(VEGFR2:G-RCFP) fish could form the basis of an in vivo assay for angiogenesis inhibitors. Zebrafish embryos arrayed in 96-well plates and subjected to a large number of different compounds have been used successfully to identify compounds that interfere with early development in the zebrafish.25 The dramatic changes in G-RCFP fluorescence that we observed after application of angiogenesis inhibitors should be easily quantifiable, and with new developments in microplate reader capabilities, it should be possible to develop a system for rapidly screening thousands of molecules per week for anti-angiogenic activity. Given the importance of this area for the identification of new drugs for cancer treatment, a simple, potentially quantitative, relatively inexpensive assay, such as that described in this letter, would be a major addition to the field of angiogenesis drug screening.
References
1. Hanahan D, Folkman J. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell. 1996; 86: 353-364.[CrossRef][Medline] [Order article via Infotrieve]
2. Li CY, Shan S, Cao Y, Dewhirst MW. Role of incipient angiogenesis in cancer metastasis. Cancer Met Rev. 2000; 19: 7–11.[Medline] [Order article via Infotrieve]
3. Carmeliet P, Jain RK. Angiogenesis in cancer and other diseases. Nature. 2000; 407: 249–257.[CrossRef][Medline] [Order article via Infotrieve]
4. Rosen L. Antiangiogenic strategies and agents in clinical trials. Oncologist. 2000; 5 (Suppl 1): 20–27.
5. Isogai S, Horiguchi M, Weinstein BM. The vascular anatomy of the developing zebrafish: an atlas of embryonic and early larval development. Dev Biol. 2001; 230: 278–301.[CrossRef][Medline] [Order article via Infotrieve]
6. Vogel AM, Weinstein BM. Studying vascular development in the zebrafish. Trends Cardiovasc Med. 2000; 10: 352–360.[CrossRef][Medline] [Order article via Infotrieve]
7. Chan J, Bayliss PE, Wood JM, Roberts TM. Dissection of angiogenic signaling in zebrafish using a chemical genetic approach. Cancer Cell. 2002; 1: 257–267.[CrossRef][Medline] [Order article via Infotrieve]
8. Serbedzija GN, Flynn E, Willett CE. Zebrafish angiogenesis: a new model for drug screening. Angiogenesis. 1999; 3: 353–359.[CrossRef][Medline] [Order article via Infotrieve]
9. Fouquet B, Weinstein BM, Serluca FC, Fishman MC. Vessel patterning in the embryo of the zebrafish: guidance by notochord. Dev Biol. 1997; 183: 37–48.[CrossRef][Medline] [Order article via Infotrieve]
10. Liao W, Bisgrove BW, Sawyer H, Hug B, Bell B, Peters K, Grunwald DJ, Stainier DY. The zebrafish gene cloche acts upstream of a flk-1 homologue to regulate endothelial cell differentiation. Development. 1997; 124: 381–389.[Abstract]
11. Weinstein BM, Stemple DL, Driever W, Fishman MC. Gridlock, a localized heritable vascular patterning defect in the zebrafish. Nat Med. 1995; 1: 1143–1147.[CrossRef][Medline] [Order article via Infotrieve]
12. Motoike T, Loughna S, Perens E, Roman BL, Liao W, Chau TC, Richardson CD, Kawate T, Kuno J, Weinstein BM, Stainier DYR, Sato TN. Universal GFP reporter for the study of vascular development. Genesis. 2000; 28: 75–81.[CrossRef][Medline] [Order article via Infotrieve]
13. Lawson ND, Weinstein BM. In vivo imaging of embryonic vascular development using transgenic zebrafish. Dev Biol. 2002; 248: 307–318.[CrossRef][Medline] [Order article via Infotrieve]
14. Matz MV, Fradkov AF, Labas YA, Savitsky AP, Zaraisky AG, Markelov ML, Lukyanov SA. Fluorescent proteins from nonbioluminescent Anthozoa species. Nat Biotech. 2000; 17: 969–973.
15. Gale NW, Yancopoulos GD. Growth factors acting via endothelial cell-specific receptor tyrosine kinases: VEGFs, angiopoietins, and ephrins in vascular development. Genes Dev. 1999; 13: 1055–1066.
16. Thompson MA, Ransom DG, Pratt SJ, MacLennan H, Kieran MW, Detrich HW, Vail B, Huber TL, Paw B, Brownlie AJ, Oates AC, Fritz A, Gates MA, Amores A, Bahary N, Talbot WS, Her H, Beier DR, Postlethwait JH, Zon LI. The cloche and spadetail genes differentially affect hematopoiesis and vasculogenesis. Dev Biol. 1998; 197: 248–269.[CrossRef][Medline] [Order article via Infotrieve]
17. Jain RK, Schlenger K, Höckel M, Yuan F. Quantitative angiogenesis assays: progress and problems. Nat Med. 1997; 3: 1203–1208.[CrossRef][Medline] [Order article via Infotrieve]
18. Fong TA, Shawver LK, Sun L, Tang C, App H, Powell TJ, Kim YH, Schreck R, Wang X, Risau W, Ullrich A, Hirth KP, McMahon G. SU5416 is a potent and selective inhibitor of the vascular endothelial growth factor receptor (Flk-1/KDR) that inhibits tyrosine kinase catalysis, tumor vascularization, and growth of multiple tumor types. Cancer Res. 1999; 59: 99–106.
19. Mendel DB, Schreck RE, West DC, Li G, Strawn LM, Tanciongco SS, Vasile S, Shawver LK, Cherrington JM. The angiogenesis inhibitor SU5416 has long-lasting effects on vascular endothelial growth factor receptor phosphorylation and function. Clin Canc Res. 2000; 6: 4848–4858.
20. Laird AD, Vajkoczy P, Shawver LK, Thurnher A, Liang C, Mohammadi M, Schlessinger J, Ullrich A, Hubbard SR, Blake RA, Fong TA, Strawn LM, Sun L, Tang C, Hawtin R, Tang F, Shenoy N, Hirth KP, McMahon G, Cherrington. JM. SU6668 is a potent antiangiogenic and antitumor agent that induces regression of established tumors. Cancer Res. 2000; 60: 4152–4160.
21. Shaheen RM, Davis DW, Liu W, Zebrowski BK, Wilson MR, Bucana CD, McConkey DJ, McMahon G, Ellis LM. Antiangiogenic therapy targeting the tyrosine kinase receptor for vascular endothelial growth factor receptor inhibits the growth of colon cancer liver metastasis and induces tumor and endothelial cell apoptosis. Cancer Res. 1999; 59: 5412–5416.
22. Mendel DB, Laird AD, Smolich BD, Blake RA, Liang C, Hannah AL, Shaheen RM, Ellis LM, Weitman S, Shawver LK, Cherrington JM. Development of SU5416, a selective small molecule inhibitor of VEGF receptor tyrosine kinase activity, as an anti-angiogenesis agent. Anticancer Drugs. 2000; 15: 29–41.
23. Nasevicius A, Larson J, Ekker SC. Distinct requirements for zebrafish angiogenesis revealed by a VEGF-A morphant. Yeast. 2000; 17: 294–301.[CrossRef][Medline] [Order article via Infotrieve]
24. Childs S, Chen J-N, Garrity DM, Fishman MC. Patterning of angiogenesis in the zebrafish embryo. Development. 2002; 129: 973–982.
25. Peterson RT, Link BA, Dowling JE, Schreiber SL. Small molecule developmental screens reveal the logic and timing of vertebrate development. Proc Natl Acad Sci U S A. 2000; 97: 12965–12969.
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