Identification of vascular disruptor compounds by analysis in zebrafish embryos and mouse embryonic endothelial cells.

To identify vascular disruptor compounds (VDCs), this study utilized an in vivo zebrafish embryo vascular model in conjunction with a mouse endothelial cell model to screen a subset of the U.S. Environmental Protection Agency (EPA) ToxCast Phase I chemical inventory. In zebrafish, 161 compounds were screened and 34 were identified by visual inspection as VDCs, of which 28 were confirmed as VDCs by quantitative image analysis. Testing of the zebrafish VDCs for their capacity to inhibit endothelial tube formation in the murine yolk-sac-derived endothelial cell line C166 identified 22 compounds that both disrupted zebrafish vascular development and murine endothelial in vitro tubulogenesis. Putative molecular targets for the VDCs were predicted using EPA's Toxicological Prioritization Index tool and a VDC signature based on a proposed adverse outcome pathway for developmental vascular toxicity. In conclusion, our screening approach identified 22 novel VDCs, some of which were active at nanomolar concentrations.

Embryonic exposure to sodium arsenite perturbs vascular development in zebrafish.

Exposure to arsenic in its inorganic form, arsenite, causes adverse effects to many different organs and tissues. Here, we have investigated arsenite-induced adverse effects on vascular tissues in the model organism zebrafish, Danio rerio. Zebrafish embryos were exposed to arsenite at different exposure windows and the susceptibility to vascular tissue damage was recorded at 72hours post fertilization (hpf). Intersegmental vessel sprouting and growth was most perturbed by exposure to arsenite during the 24-48hpf window, while disruption in the condensation of the caudal vein plexus was more often observed at the 48-72hpf exposure window, reflecting when these structures develop during normal embryogenesis. The vascular growth rate was decreased by arsenite exposure, and deviated from that of control embryos at around 24-26.5hpf. We further mapped changes in expression of key regulators of angiogenesis and vasculogenesis. Downregulation of vascular endothelial growth factor receptor 1/fms-related tyrosine kinase 1 (vegfr1/flt1) expression was evident already at 24hpf, coinciding with the decreased vascular growth rate. At later time points, matrix metalloproteinase 9 (mmp9) expression was upregulated, suggesting that arsenite affects the composition of the extracellular matrix. In total, the expression of eight key factors involved in different aspects of vascularization was significantly altered by arsenic exposure. In conclusion, our results show that arsenite is a potent vascular disruptor in the developing zebrafish embryo, a finding that calls for an evaluation of arsenite as a developmental vascular toxicant in mammalian model systems.

Automated analysis of zebrafish images for screening toxicants.

An important factor facilitating the application of zebrafish in biomedical research is high throughput screening of vertebrate animal models. For example, being able to model the growth of blood vessel in the vasculature system is interesting for understanding both the circulatory system in humans, and for facilitating large scale screening of the influence of various chemicals on vascular development. Compared to other models, the zebrafish embryo is an attractive alternative for environmental risk assessment of chemicals since it offers the possibility to perform high-throughput analyses in vivo. However the lack of an automated image analysis framework restricts high throughput screening. In this paper, we provide a method for quantitative measurements of zebrafish blood vessel morphology since it is difficult to assess changes in vessel structure by visual inspection. The method presented is generalized, i.e. it is not restricted to any specific chemically treated zebrafish, and can be used with wide variety of chemicals.

3D imaging for quantitative assessment of toxicity on vascular development in zebrafish.

In this study, we describe the utility of the zebrafish model of in-vivo blood vessel formation as a tool for chemical risk assessment. Time-lapse confocal imaging of embryonic vasculature in the zebrafish is used in conjunction with digital image analysis to monitor and quantify the effect of toxins on vascular development. Non-rigid registration is used to capture changes in vascular morphology over time. Vascular formation in healthy normal and arsenic treated embryos was evaluated for differences in vascular structure using the algorithms developed. Although, the temporal progression of vascular development was similar, significant differences were observed in vessel structure between the toxin treated and healthy fish. This study revealed, for the first time, that vital vascular structures in fish maybe affected by exposure to arsenic. This technique allowed visualization of vascular abnormalities in embryos showing no external signs of malformations.