Current methods for assaying angiogenesis in vitro and in vivo

Abstract

Angiogenesis, the development of new blood vessels from an existing vasculature, is essential in normal developmental processes and in numerous pathologies, including diabetic retinopathy, psoriasis and tumour growth and metastases. One of the problems faced by angiogenesis researchers has been the difficulty of finding suitable methods for assessing the effects of regulators of the angiogenic response. The ideal assay would be reliable, technically straightforward, easily quantifiable and, most importantly, physiologically relevant. Here, we review the advantages and limitations of the principal assays in use, including those for the proliferation, migration and differentiation of endothelial cells in vitro, vessel outgrowth from organ cultures and in vivo assays such as sponge implantation, corneal, chamber, zebrafish, chick chorioallantoic membrane (CAM) and tumour angiogenesis models.

Figure 1

Migration of human dermal microvascular endothelial cells (HuDMECs) in Boyden chamber in response to basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). (a) Appearance of HuDMEC (haematoxylin stained) on the underside of the membrane in the migration assay. Cells migrating in response to (I) control (II) bFGF 10 ng/ml and (III) VEGF 10 ng/ml (×160 magnification). (b) The increase in migration of HuDMECs in response to increasing concentrations of each growth factor.

Figure 2

Appearance of human dermal microvascular endothelial cells (HuDMECs) forming tubules in the Matrigel assay. Tubule formation in response to (a) control, (b) basic fibroblast growth factor (10 ng/ml) and (c) vascular endothelial growth factor (10 ng/ml) (×40 magnification).

Figure 3

Formation of tubules from an aortic ring. The aortic ring is cultured in full growth medium containing vascular endothelial growth factor. The appearance of tubules can clearly be seen. Photograph courtesy of Dr Roy Bicknell, CRUK Molecular Angiogenesis Laboratory, Oxford.

Figure 4

The development of new vasculature in the chick chorioallantoic membrane (CAM) assay. The appearance of the CAM in the absence (a) or presence (b) of thymidine phosphorylase. Photographs courtesy of Dr Roy Bicknell, CRUK, Oxford.

Figure 5

The dorsal skinfold window chamber model. After recovery from surgery, the neovascularization in the chamber is imaged using in vivo microscopy. Scale bar, 15 mm.

Figure 6

Angiogenesis in the zebrafish embryo. Angiogram of a zebrafish larva at 7 day post fertilization (lateral view, anterior to the left, dorsal side up). The zebrafish fli 1 promoter was used to drive the expression of enhanced green fluorescent protein (EGFP) in all blood vessels; continuous in vivo observation of the vertebrate embryonic vasculature was achieved by use of time-lapse multiphoton laser microscopy. Scale bar, 500 µm (Image courtesy of Dr BM Weinstein, Unit on Vertebrate Organogegesis, Laboratory of Molecular Genetics, NICHD, Bethesda, MD).