Rapid analysis of angiogenesis drugs in a live fluorescent zebrafish assay.

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 …

[1]  D. Harrison,et al.  Superoxide production, risk factors, and endothelium-dependent relaxations in human internal mammary arteries. , 1999, Circulation.

[2]  G. Serbedzija,et al.  Zebrafish angiogenesis: A new model for drug screening , 2004, Angiogenesis.

[3]  D. Hanahan,et al.  Patterns and Emerging Mechanisms of the Angiogenic Switch during Tumorigenesis , 1996, Cell.

[4]  K Y Liang,et al.  Longitudinal data analysis for discrete and continuous outcomes. , 1986, Biometrics.

[5]  J. Wood,et al.  Dissection of angiogenic signaling in zebrafish using a chemical genetic approach. , 2002, Cancer cell.

[6]  D. Stainier,et al.  The zebrafish gene cloche acts upstream of a flk-1 homologue to regulate endothelial cell differentiation. , 1997, Development.

[7]  Leslie A. Smith,et al.  Long-Term Vitamin C Treatment Increases Vascular Tetrahydrobiopterin Levels and Nitric Oxide Synthase Activity , 2003, Circulation research.

[8]  J. Cherrington,et al.  The angiogenesis inhibitor SU5416 has long-lasting effects on vascular endothelial growth factor receptor phosphorylation and function. , 2000, Clinical cancer research : an official journal of the American Association for Cancer Research.

[9]  S. Milstien,et al.  Regulation of nitric oxide synthesis by proinflammatory cytokines in human umbilical vein endothelial cells. Elevations in tetrahydrobiopterin levels enhance endothelial nitric oxide synthase specific activity. , 1994, The Journal of clinical investigation.

[10]  Wolfgang Driever,et al.  gridlock, a localized heritable vascular patterning defect in the zebrafish , 1995, Nature Medicine.

[11]  References , 1971 .

[12]  Rakesh K. Jain,et al.  Quantitative angiogenesis assays: Progress and problems , 1997, Nature Medicine.

[13]  F. Karpe,et al.  Variants of the microsomal triglyceride transfer protein gene are associated with plasma cholesterol levels and body mass index. , 2002, Journal of lipid research.

[14]  S. Zeger,et al.  Longitudinal data analysis using generalized linear models , 1986 .

[15]  Cherrington,et al.  SU6668 is a potent antiangiogenic and antitumor agent that induces regression of established tumors. , 2000, Cancer research.

[16]  L. Ellis,et al.  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. , 1999, Cancer research.

[17]  S. Rajagopalan,et al.  Altered Tetrahydrobiopterin Metabolism in Atherosclerosis: Implications for Use of Oxidized Tetrahydrobiopterin Analogues and Thiol Antioxidants , 2002, Arteriosclerosis, thrombosis, and vascular biology.

[18]  B. Weinstein,et al.  In vivo imaging of embryonic vascular development using transgenic zebrafish. , 2002, Developmental biology.

[19]  S. Juo,et al.  Common polymorphism in promoter of microsomal triglyceride transfer protein gene influences cholesterol, ApoB, and triglyceride levels in young african american men: results from the coronary artery risk development in young adults (CARDIA) study. , 2000, Arteriosclerosis, thrombosis, and vascular biology.

[20]  P. Wilson,et al.  Absence of association between genetic variation in the promoter of the microsomal triglyceride transfer protein gene and plasma lipoproteins in the Framingham Offspring Study. , 2000, Atherosclerosis.

[21]  P. Carmeliet,et al.  Angiogenesis in cancer and other diseases , 2000, Nature.

[22]  L. Ellis,et al.  Development of SU5416, a selective small molecule inhibitor of VEGF receptor tyrosine kinase activity, as an anti-angiogenesis agent. , 2000, Anti-cancer drug design.

[23]  O. Augusto,et al.  Peroxynitrite-mediated formation of free radicals in human plasma: EPR detection of ascorbyl, albumin-thiyl and uric acid-derived free radicals. , 1996, The Biochemical journal.

[24]  M. Dewhirst,et al.  Role of Incipient Angiogenesis in Cancer Metastasis , 2004, Cancer and Metastasis Reviews.

[25]  B. Weinstein,et al.  Studying vascular development in the zebrafish. , 2000, Trends in cardiovascular medicine.

[26]  S. Ekker,et al.  Distinct requirements for zebrafish angiogenesis revealed by a VEGF-A morphant. , 2000, Yeast.

[27]  J. Dowling,et al.  Small molecule developmental screens reveal the logic and timing of vertebrate development. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[28]  A. Amores,et al.  The cloche and spadetail genes differentially affect hematopoiesis and vasculogenesis. , 1998, Developmental biology.

[29]  N. Blau,et al.  Mutations in the sepiapterin reductase gene cause a novel tetrahydrobiopterin-dependent monoamine-neurotransmitter deficiency without hyperphenylalaninemia. , 2001, American journal of human genetics.

[30]  T. Fukushima,et al.  Analysis of reduced forms of biopterin in biological tissues and fluids. , 1980, Analytical biochemistry.

[31]  J. Joseph,et al.  The ratio between tetrahydrobiopterin and oxidized tetrahydrobiopterin analogues controls superoxide release from endothelial nitric oxide synthase: an EPR spin trapping study. , 2002, The Biochemical journal.

[32]  N. Blau,et al.  Reduced nitric oxide metabolites in CSF of patients with tetrahydrobiopterin deficiency , 2002, Journal of neurochemistry.

[33]  A. Ullrich,et al.  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. , 1999, Cancer research.

[34]  B. Weinstein,et al.  The vascular anatomy of the developing zebrafish: an atlas of embryonic and early larval development. , 2001, Developmental biology.

[35]  M. Fishman,et al.  Vessel patterning in the embryo of the zebrafish: guidance by notochord. , 1997, Developmental biology.

[36]  S. Lukyanov,et al.  Fluorescent proteins from nonbioluminescent Anthozoa species , 1999, Nature Biotechnology.

[37]  L. Rosen,et al.  Antiangiogenic strategies and agents in clinical trials. , 2000, The oncologist.

[38]  N. Blau,et al.  Detection of sepiapterin in CSF of patients with sepiapterin reductase deficiency. , 2002, Molecular genetics and metabolism.

[39]  A. Hamsten,et al.  A common functional polymorphism in the promoter region of the microsomal triglyceride transfer protein gene influences plasma LDL levels. , 1998, Arteriosclerosis, thrombosis, and vascular biology.

[40]  G. Yancopoulos,et al.  Growth factors acting via endothelial cell-specific receptor tyrosine kinases: VEGFs, angiopoietins, and ephrins in vascular development. , 1999, Genes & development.

[41]  Thomas N. Sato,et al.  Universal GFP reporter for the study of vascular development , 2000, Genesis.

[42]  M. Fishman,et al.  Patterning of angiogenesis in the zebrafish embryo. , 2002, Development.