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Review
. 2009 Jun;90(3):195-221.
doi: 10.1111/j.1365-2613.2008.00633.x.

A critical analysis of current in vitro and in vivo angiogenesis assays

Carolyn A Staton  1 , Malcolm W R Reed, Nicola J Brown
Affiliations
Review

A critical analysis of current in vitro and in vivo angiogenesis assays

Carolyn A Staton et al. Int J Exp Pathol. 2009 Jun.

Abstract

The study of angiogenesis has grown exponentially over the past 40 years with the recognition that angiogenesis is essential for numerous pathologies and, more recently, with the advent of successful drugs to inhibit angiogenesis in tumours. The main problem with angiogenesis research remains the choice of appropriate assays to evaluate the efficacy of potential new drugs and to identify potential targets within the angiogenic process. This selection is made more complex by the recognition that heterogeneity occurs, not only within the endothelial cells themselves, but also within the specific microenvironment to be studied. Thus, it is essential to choose the assay conditions and cell types that most closely resemble the angiogenic disease being studied. This is especially important when aiming to translate data from in vitro to in vivo and from preclinical to the clinic. Here we critically review and highlight recent advances in the principle assays in common use including those for endothelial cell proliferation, migration, differentiation and co-culture with fibroblasts and mural cells in vitro, vessel outgrowth from organ cultures and in vivo assays such as chick chorioallantoic membrane (CAM), zebrafish, sponge implantation, corneal, dorsal air sac, chamber and tumour angiogenesis models. Finally, we briefly discuss the direction likely to be taken in future studies, which include the use of increasingly sophisticated imaging analysis systems for data acquisition.

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Figures

Figure 1
Figure 1
Endothelial cells. Representative photographs of confluent human umbilical vein endothelial cells (HUVEC; a–c) and human dermal microvascular endothelial cells (HuDMEC; d–f) as seen with phase contrast microscopy (a, d), or stained with CD31 (b, e) or von Willebrand’s Factor (c, f). In the fluorescent photographs, the nuclei are counterstained with DAPI. The morphology of both cell types is very similar although the HUVEC form a more classical cobblestone pattern. Photographs courtesy of Promocell GmbH, Heidelberg, Germany.
Figure 2
Figure 2
‘Wound healing’ migration assay. The pictures show HuDMEC cells that have been (a) freshly wounded and (b) six hours after wounding at which point cell migration into the area of the wound can clearly be seen. Dotted lines mark the edge of the ‘wound’. Photographs courtesy of Chryso Kanthou, University of Sheffield, UK.
Figure 3
Figure 3
Tubule formation assays. (a) Appearance of HuDMEC forming tubules when plated onto GF-reduced Matrigel in the absence (i) or presence (ii) of VEGF (10 ng/ml). (b) Appearance of tubules complete with lumen (star) formed by endothelial cells on microcarrier beads in a fibrin gel.
Figure 4
Figure 4
Formation of tubules from a human umbilical artery. The umbilical artery is cultured in full growth medium and the appearance of tubules sprouting out from the artery can clearly be seen (ringed by a dotted line).
Figure 5
Figure 5
Angiogenesis in the zebrafish embryo. A confocal image of a Fli-eGFP zebrafish embryo showing green blood vessels at 48 h postfertilization. dsRED microspheres were injected into the sinus venosus allowing time lapse confocal laser imaging along with digital motion analysis to track the passage of the dsRED microspheres through the zebrafish circulation. Scale bar, 200 μm. A, anterior; ISV, intersegmental vessels; P, posterior; SIV, subintestinal veins; SV, sinus venosus; Y, yolksac. Image courtesy of Russell Hughes, Philip Ingham & Claire Lewis, University of Sheffield, UK.
Figure 6
Figure 6
The dorsal skinfold chamber assay. Neovascularization can be observed in the chamber using in vivo microscopy.
Figure 7
Figure 7
Visualization of tumours growing in orthotopic sites. MDA-MB-436 breast cancer cells transfected to express GFP were injected into the mammary fat pad and can be observed using fluorescent microscopy.
Figure 8
Figure 8
eNOS-GFP vessels. A confocal image of green fluorescent blood vessels, which are clearly visible by virtue of the eNOS expression at the endothelial cell barrier within the eNOS-GFP transgenic mice.
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