Skip to main content

Advertisement

SpringerLink
Log in

Use of a monoclonal antibody specific for activated endothelial cells to quantitate angiogenesis in vivo in zebrafish after drug treatment

  • Published:
Angiogenesis Aims and scope Submit manuscript

Abstract

We have recently generated a monoclonal antibody (mAb), Phy-V002, which specifically labels activated vascular endothelial cells (EC) in zebrafish. Here, we show that this mAb labels activated EC in newly formed vessels in vivo without staining mature vessels or other tissues. Using this mAb, drug effects on in vivo EC migration and vessel formation were visually assessed by whole-mount immunochemical staining in the transparent embryo. In addition, we have developed a quantitative microplate-based ELISA that measures EC proliferation in vivo after drug treatment. We have validated the quantitative in vivo ELISA using several antiangiogenic small molecules with different mechanisms of action which were added directly to the fish water. Some of these drugs, including: 2-methoxyestradiol, flavopiridol, paclitaxel, and genistein, are currently in clinical trials. We also injected large molecule drugs, including 3TSR and TSR2+KRFK, recombinant human antiangiogenic peptides of thrombospondin-1, a natural protein. To demonstrate that proangiogenic effects can also be assessed in zebrafish, we assessed effects of penicillamine and simvastatin, two proangiogenic compounds shown to stimulate vessel formation in rodents. Using whole-mount immunochemical staining with Phy-V002, inhibition of EC migration and inhibition or stimulation of vessel formation were visually observed for each compound. Next, using the quantitative in vivo angiogenesis ELISA, we generated dose-response curves for each compound. Compared to conventional assays, advantages of using zebrafish to assess drug effects on angiogenesis include: (1) a short assay time; (2) easy animal maintenance; (3) use of small quantities of drug; (4) single dosing; (5) a quantitative assay format; and (6) use of statistically significant number of animals per test.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

AA:

branchial arch vessel

BSA:

bovine serum albumin

CCV:

common cardinal vein

CV:

cranial vessel

CVP:

caudal vessel plexus

DA:

dorsal aorta

DLAV:

dorsal longitudinal anastomotic vessel

DMSOD2:

0.8 DMSO treated 2 dpf embryo

dpf:

days post fertilization

EC:

endothelial cell

hpf:

hours post fertilization

hpi:

hours post injection

HRP:

horseradish peroxidase

mAb:

monoclonal antibody

ISV:

intersegmental vessel

2-ME:

2 Methoxyestradiol

NC:

notochord

ND2:

untreated normal 2 dpf embryo

PAV:

parachordal vessel

PBS:

phosphate buffered saline, pH 7.0

PBST:

PBS containing 0.1 Tween-20

PCV:

posterior cardinal vein

RBC:

red blood cell

WBC:

white blood cell

S:

somite

SIV:

subintestinal vessel

TSP-1:

thrombospondin-1

3TSR:

3 type-1 repeats peptide fragment of TSP-1

TSR2 + KRFK:

2nd type-1 repeat plus KRFK motif peptide fragment of TSP-1

T3C1:

type-3 domain plus C terminal peptide fragment of TSP-1

VV:

ventral vein

References

  • F Shalaby et al. (1995) ArticleTitleFailure of blood island formation and vasculogenesis in Flk-1-deficient mice Nature 376 62–66

    Google Scholar 

  • B Fouquet et al. (1997) ArticleTitleVessel patterning in the embryo of the zebrafish: guidance by notochord Dev Biol 183 37–48

    Google Scholar 

  • M Gehring et al. (1998) ArticleTitleThe SCL gene specifies haemangioblast development form early mesoderm EMBO J 17 4029–4045

    Google Scholar 

  • W Liao et al. (1997) ArticleTitleThe zebrafish gene cloche acts upstream of a flk-1 homologue to regulate endothelial cell differentiation Development 124 381–389

    Google Scholar 

  • MS Lyons et al. (1998) ArticleTitleIsolation of the zebrafish homologues for the tie-1 and tie-2 endothelium-specific receptor tyrosine kinases Dev Dyn 212 133–140

    Google Scholar 

  • D Hanahan (1997) ArticleTitleSignaling vascular morphogenesis and maintenance Science 277 48–50

    Google Scholar 

  • D Liang et al. (1998) ArticleTitleCloning and characterization of vascular endothelial growth factor (VEGF) from zebrafish Danio rerio Biochimica et Biophysica Acta 1397 14–20

    Google Scholar 

  • M Westerfield (1993) The Zebrafish Book: A Guide for the Laboratory use of Zebrafish The University of Oregon Press Eugene

    Google Scholar 

  • S Childs et al. (2002) ArticleTitlePatterning of angiogenesis in the zebrafish embryo Development 129 973–82

    Google Scholar 

  • S Isogai M Horiguchi BM Weinstein (2001) ArticleTitleThe vascular anatomy of the developing zebrafish: an atlas of embryonic and early larval development Dev Biol 230 278–301

    Google Scholar 

  • RM Shaheen et al. (1999) ArticleTitleAntiangiogenic 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 59 5412–6

    Google Scholar 

  • N Mabjeesh et al. (2003) ArticleTitle2ME2 inhibits tumor growth and angiogenesis by disrupting microtubules and dysregulating HIF Cancer Cell 3 IssueID4 363–75

    Google Scholar 

  • S Mooberry (2003) ArticleTitleMechanism of action of 2-methoxyestradiol: new developments Drug Resist Updat 6 IssueID6 355–61

    Google Scholar 

  • W Shang I Konidari D Schomberg (2001) ArticleTitle2-Methoxyestradiol an endogenous estradiol metabolite differentially inhibits granulosa and endothelial cell mitosis: a potential follicular antiangiogenic regulator Biol Reprod 65 622–7

    Google Scholar 

  • A Tan S Swain (2002) ArticleTitleReview of flavopiridol a cyclin-dependent kinase inhibitor as breast cancer therapy Semin Oncol 29 IssueID3 Suppl 11 77–85

    Google Scholar 

  • S Zhai et al. (2002) ArticleTitleFlavopiridol a novel cyclin-dependent kinase inhibitor, in clinical development Ann Pharmacother 36 IssueID5 905–11

    Google Scholar 

  • P Albertsson Lennernas K Norrby (2003) ArticleTitleChemotherapy and antiangiogenesis: Drug-specific effects on microvessel sprouting APMIS 111 IssueID11 995–1003

    Google Scholar 

  • K Klos et al. (2003) ArticleTitleCombined trastuzumab and paclitaxel treatment better inhibits ErbB-2-mediated angiogenesis in breast carcinoma through a more effective inhibiton of Akt than either treatment alone Cancer 98 IssueID7 1377–85

    Google Scholar 

  • S Ng W Figg A Sparreboom (2004) ArticleTitleTaxane-mediated antiangiogenesis in vitro: influence of formulation vehicles and binding proteins Cancer Res 64 821–4

    Google Scholar 

  • P Buchler et al. (2004) ArticleTitleantiangiogenic activity of genistein in pancreatic carcinoma cell is mediated by the inhibition of hypoxia-inducible factor-1 and the down regulation of VEGF gene expression Cancer 100 IssueID1 201–10

    Google Scholar 

  • C McDonnell et al. (2004) ArticleTitleImprovement in efficacy of chemoradiotherapy by addition of an antiangiogenic agent in a murine tumor model J Surg Res 116 IssueID1 19–23

    Google Scholar 

  • M Pepper et al. (2004) ArticleTitle8-prenylnaringenin a novel phytoestrogen inhibits angiogenesis in vitro and in vivo J Cell Physiol 199 IssueID1 98–107

    Google Scholar 

  • WM Miao et al. (2001) ArticleTitleThrombospondin-1 type 1 repeat recombinant proteins inhibit tumor growth through transforming growth factor-beta-dependent and -independent mechanisms Cancer Res 61 IssueID21 7830–9

    Google Scholar 

  • A Brouet et al. (2001) ArticleTitleHsp90 and caveolin are key targets for the proangiogenic nitric oxide-mediated effects of statins Circ Res 89 866–73

    Google Scholar 

  • K Kawasaki et al. (2003) ArticleTitleActivation of the phosphatidylinositol 3-kinase/protein kinase Akt pathway mediates nitric oxide-induced endothelial cell migration and angiogenesis Mol Cell Biol 23 IssueID16 5726–37

    Google Scholar 

  • I Pourati et al. (2003) ArticleTitleStatin use is associated with enhanced collateralization of severely diseased coronary arteries Am Heart J 146 IssueID5 876–81

    Google Scholar 

  • MHC Weis A Glassford J Cooke (2002) ArticleTitleStatins have biphasic effects on angiogenesis Circulation 105 739–45

    Google Scholar 

  • S Gilbertson-Beadling et al. (1995) ArticleTitleThe tetracycline analogs minocycline and doxycycline inhibit angiogenesis in vitro by a non-metalloproteinase-dependent mechanism Cancer Chemother Pharmacol 36 IssueID5 418–24

    Google Scholar 

  • N Klauber et al. (1997) ArticleTitleInhibition of angiogenesis and breast cancer in mice by the microtubule inhibitors 2-methoxyestradiol and taxol Cancer Res 57 IssueID1 81–6

    Google Scholar 

  • H Kohkichi et al. (2004) ArticleTitleEvaluation of the antiangiogenic effect of Taxol in a human epithelial ovarian carcinoma cell line Cancer Chemother Pharmacol 53 68–74

    Google Scholar 

  • T Nishishita P Lin (2004) ArticleTitleAngiopoietin 1, PDGF-B, and TGF-beta gene regulation in endothelial cell and smooth muscle cell interaction J Cell Biochem 91 IssueID3 584–93

    Google Scholar 

  • ND Lawson BM Weinstein (2002) ArticleTitleIn vivo imaging of embryonic vascular development using transgenic zebrafish Dev Biol 248 IssueID2 307–18

    Google Scholar 

  • T Motoike et al. (2000) ArticleTitleUniversal GFP reporter for the study of vascular development Genesis 28 IssueID2 75–81

    Google Scholar 

  • L Parker et al. (2004) ArticleTitleThe endothelial-cell-derived secreted factor Egfl7 regulates vascular tube formation Nature 428 754

    Google Scholar 

  • CE Willett et al. (2001) ArticleTitleIkaros expression as a marker for lymphoid progenitors during zebrafish development Dev Dyn 222 IssueID4 694–8

    Google Scholar 

  • MA Thompson et al. (1998) ArticleTitleThe cloche and spadetail genes differentially affect hematopoiesis and vasculogenesis Dev Biol 197 248–69

    Google Scholar 

  • O Gallo et al. (2002) ArticleTitleCorrelation between nitric oxide and cyclooxygenase-2 pathway in head and neck squamous cell carcinomas Biochem Biophys Res Commun 299 IssueID4 517–24

    Google Scholar 

  • A Jozkowicz et al. (2001) ArticleTitleGenetic augumentation of nitric oxide synthase increases the vascular generation of VEGF Cardiovasc Res 51 IssueID4 773–83

    Google Scholar 

Download references

Author information

Author notes

  1. Jenny Lee

    Present address: Oncogene Science/Bayer Corporation, Cambridge, Massachusetts, USA

Authors and Affiliations

  1. Phylonix Pharmaceuticals, Inc., 100 Inman Street, suite #300, Cambridge, MA, 02139, USA

    Wen Lin Seng, Kurt Eng & Patricia McGrath

Authors
  1. Wen Lin Seng
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kurt Eng
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jenny Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Patricia McGrath
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Wen Lin Seng.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Seng, W.L., Eng, K., Lee, J. et al. Use of a monoclonal antibody specific for activated endothelial cells to quantitate angiogenesis in vivo in zebrafish after drug treatment. Angiogenesis 7, 243–253 (2004). https://doi.org/10.1007/s10456-004-4181-7

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10456-004-4181-7

Keywords

Search

Navigation