We have generated a monoclonal antibody (mAb Phy-V002) that specifically labels activated endothelial cells (EC) in zebrafish. Based on the specificity of the mAb, we have developed a zebrafish ELISA that measures EC proliferation in vivo, which is suitable for high throughput screening for angiogenesis modulators. In addition, drug effects on EC migration and vessel formation in vivo can be confirmed visually by whole mount immunochemical staining of the transparent zebrafish with this mAb. We have validated the assays using a number of anti-angiogenic compounds with different mechanisms of action, including SU5416, Flavopiridol, Genistein, 2-Me, and Paclitaxel. Several human recombinant peptides (including 3TSR and TSR2+KRFK) were also tested by microinjection, and showed inhibition of zebrafish EC proliferation. Inhibitory effects in vessel formation in zebrafish and mouse tumor models were comparable.

Compared to conventional assays, advantages of the in vivo zebrafish angiogenesis ELISA include: 1) short assay time; 2) easy animal maintenance; 3) use of small quantity of drug; 4) single dosing; 5) quantitative assay format; and 6) statistically significant number of animals per test. These advantages make the in vivo zebrafish angiogenesis ELISA an ideal high throughput format for in vivo angiogenesis drug screening.

The angiogenic vessels in zebrafish can be detected with mAb Phy-V002, which specifically labels activated endothelial cells (EC) in zebrafish.

Zebrafish were stained with activated endothelial cell (EC) specific mAb. Left panel shows several vessels forming in the head region (arrows). A, B, C show the white squares in the left panel at higher magnification. D is an image of a single intersegmental vessel (ISV) in the trunk. At higher magnification, individual cells are identifiable. Staining is strongest in the dividing and budding regions of the cells (A, B, C, D).

Quantitative in vivo EC proliferation ELISA assay

Drug-treated and control zebrafish are processed for whole animal ELISA using the angiogenic vessel-specific antibody Phy-V002 as the primary antibody and HRP as the reporter enzyme. Sixty zebrafish are treated with each concentration of each compound. Drug effects are expressed as % inhibition of EC proliferation (compared to negative control). Concentration-response curves are generated for assays with multiple concentrations.

Dose-response curve showing effects of 2-ME
on inhibition of EC proliferation.

Dose-response curve showing effects of Flavopiridol
on inhibition of EC proliferation.

Visual assessment of drug effects on in vivo EC migration

The transparency of zebrafish and the specificity of Phy-V002 enable assessment of in vivo EC migration, which is impossible in other angiogenesis assays. Twenty zebrafish are treated with each concentration of each compound. Compound-treated and control zebrafish are processed for whole mount staining with Phy-V002 and Rhodomine-conjugated secondary antibody. For each treatment condition, 10 zebrafish are randomly selected for visual assessment of the whole embryo to assess inhibition in EC migration. Embryos are examined at high magnification to detect abnormal aggregation of ECs.

EC migration defects detected by Phy-V002 staining. (A), control, showing normal distribution of ECs in ISVs (red arrow) and caudal vessel plexus (yellow arrows). (B), zebrafish treated with Tetracycline. Note the abnormal aggregation of ECs outside the vasculature (white arrow).

Vessel inhibition assay

I. Visual assessment of drug effects on in vivo vessel formation

Twenty zebrafish are treated with each concentration of each compound. Compound-treated and control zebrafish are processed for whole mount staining with Phy-V002 and Rhodomine-conjugated secondary antibody. For each treatment condition, 10 zebrafish are randomly selected for visual assessment to assess inhibition of vessel formation.

Phy-V002 whole mount staining showing disruption of intersegmental vessels (ISVs) by anti-angiogenic drug treatment. (A), control; the arrow points to one of the ISVs. (B) to (D), zebrafish treated with the following anti-angiogenic drugs: (B) SU-5416, a vascular endothelial growth factor receptor (VEGFR) kinase inhibitor; (C), Flavopiridol, a cyclin-dependent kinase inhibitor; (D), Genistein, a protein tyrosine kinase inhibitor. Note the significant reduction of ISVs after treatment with these kinase inhibitors.

II. Morphometric analysis of drug effects on vessel formation in vivo

Whole mount staining results from Part I above are quantitated by image analysis. The intersegmental vessels (ISVs) will be quantified in the trunk region in 10 zebrafish for each compound at each condition.

III. Assessment of drug effects on vessel function by microangiography

Twenty zebrafish are treated with each concentration of each compound. Compound-treated and control zebrafish are processed for microinjection of fluorescent microspheres into the circulatory system, and the integrity of the vessels and circulation are assessed.

Zebrafish functional blood vessels labeled by microangiography. (A), Normal zebrafish. (B), Zebrafish treated with SU5416. Note the significant reduction of ISVs after SU5416 treatment.