Assay | References | Advantages | Limitations |
---|---|---|---|
In vitro | |||
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) |
Ex vivo | |||
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) |
In vivo | |||
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 |