Table 1 Available assays to evaluate angiogenic potential

Endothelial proliferation assays

Gomez and Reich 2003 [19]

Yu et al. 2004 [20]

• Reproducible and easy to set up

• Provide quantifiable data

• Measure proliferation and apoptosis

• Short window of analysis after culture due to endothelial cell senescence

• Sensitive to cell density

• Involve endothelial cells from macrovascular origin (HUVEC)

Endothelial cell migration assays

• Transwell systems

• Wound healing assays

Wong and Gotlieb 1984 [21]

Schor et al. 2001 [22]

Albini et al. 2004 [23]

• Reproducible

• Short duration (4–6 hours)

• Quantitative analysis of endothelial cell migration over time

• Sensitive to small alterations in concentration gradients

• Difficult to define and maintain transmembrane gradients

• Inability to observe cell migration in transwell

• Challenging to establish matching conditions between control and experimental groups

• Difficult to obtain accurate results with low cell counts

Endothelial tube formation assays

Lawley and Kubota 1989 [24]

Kanzawa et al. 1993 [25]

Auerbach et al. 2003 [26]

• Useful to test angiogenic and anti-angiogenic effects of compounds

• More representative of in vivo angiogenesis than 2D assays

• Introduce ECM to culture system

• Measure proliferation and differentiation

• Lack of consistent lumen formation

• Homogeneous tubules

• Sensitive to uneven matrix coating of wells and cell density

• Time-consuming analysis (multiple parameters of analysis)

Chick aortic arch model

Staton et al. 2009 [18]

• System includes nonendothelial cells (pericytes, smooth muscle cells) and ECM

• Easy set up, early cell outgrowth (48 hours)

• Embryonic endothelial cells resemble microvascular phenotype

• Endothelial cells are in a proliferative state in the embryo (not representative of true in vivo scenarios)

Chick chorioallantoic membrane assay

Ribbati et al. 1995 [27]

Ejaz et al. 2004 [28]

• Simple and inexpensive

• Ideal to implant tissue or organ grafts

• CAM membrane is immunoprivileged enabling xeno-graft studies

• Ideal for large-scale screening

• Noninvasive observation

• CAM has endogenous vasculature, difficult to distinguish pre-existing and newly formed vasculature

• 11-day incubation time prior to implantation of test reagents

• Incision in shell may induce inflammation and a specific angiogenic response

• Sensitive to oxygen tension

Matrigel™ plug assay

Passaniti et al. 1992 [29]

Baker et al. 2006 [30]

• Quantitative histological analysis

• Matrigel™ provides natural environment for angiogenesis

• Time consuming, including 2 weeks of plug incubation in host, isolation, sectioning, analysis

• Costly

Sponge/matrix implant assay

Salvatore et al. 1961 [31]

Dellian et al. 1996 [32]

• Include defined polymers to study angiogenesis

• Angiogenic response can be monitored over time in live animals

• Ideal for studying tumor-induced angiogenesis

• Implants can become encapsulated with cytokine-secreting macrophages

• Inflammatory response may interfere with angiogenic response

• Undesirable fibrosis

• Variable retention of test compounds in different substrates

Corneal assay

Gimbrone et al. 1974 [33]

Muthukkaruppan and Auerbach 1979 [34]

• New blood vessels easily observed due to the absence of background blood vessels

• Executed in mice, rats, rabbits

• Noninvasive monitoring and data easily quantifiable

• Challenging surgical procedure

• Limited space of test substance injection

• Inflammation difficult to avoid

• Atypical angiogenesis because cornea is avascular

• Costly

Dorsal air sac model

Selye 1953 [35]

Oikawa et al. 1997 [36]

• Adaptable to various applications

• Allow continuous non invasive monitoring of endothelial networks

• Difficult to distinguish pre-existing and newly formed vasculature

• Delicate procedure (irritation to dorsal skin)

Zebrafish assay

Rubinstein 2003 [37]

Isogai et al. 2001 [38]

• Enables large-scale projects

• Shared angiogenic genes and mechanisms

• Easily monitored/quantified

• Ideal for testing of anti-angiogenic compounds

• Suitable for genetic studies of angiogenesis

• Relevance of fish endothelial cell angiogenesis is under debate

• Nonmammalian cell types and involves embryonic cells

• Costly to maintain breeding conditions