Angiogenesis assays represent an indispensable tool for obtaining insights into the mechanisms of angiogenesis, but also open the door for the development of various treatment strategies to modulate formation of new blood vessels. In vitro angiogenesis assays are based on the principle that endothelial cells have the ability to form three-dimensional tube structures when cultured on a suitable matrix.
Assays that involve Matrigel (which is matrix derived from murine tumors) and a co-culture of endothelial cells with fibroblasts are among most commonly employed in vitro tube formation assays. Various angiogenic inducers and inhibitors can either promote or inhibit tube formation, which is thoroughly researched.
Promoters of tube formation
Factors that can promote endothelial tube formation are also potent angiogenic factors such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), vascular endothelial growth factor receptor-1 (VEGFR1), transforming growth factor beta (TGFB), as well as platelet-derived growth factor family (PDGF).
Mentioned factors are available for purchase to use endothelial cell tube formation assay from company Thermo Fisher Scientific. For example, VEGF recombinant mouse and rat protein, FLT (VEGFR1) recombinant human protein, FGF basic recombinant human protein, TGFB1 recombinant human protein and PDGF-BB recombinant mouse protein can be employed as angiogenic inducers for research purposes.
Reduced growth factor basement membrane extract (RGF BME) is often used for testing compounds that promote angiogenesis, as there is significantly less tube formation in comparison to complete basement membrane matrix (or non-reduced BME). For example, Trevigen’s Tube Formation Kit from AMSBIO contains RGF BME that shows decreased background tube formation when angiogenic factors are absent.
Short exposure of human microvascular endothelial cells on Matrigel to hypoxia (oxygen deficiency) followed by reoxygenation enhances the formation of a network of cappilary-like tubes by a mechanism which seemingly does not involve activation of the “classical” promotors of angiogenesis.
Inhibitors of tube formation
Several studies have shown that endothelial cell tube formation can be inhibited by interferon both in vitro and in vivo, and recent results have confirmed that interferon-α substantially inhibits human umbilical vein endothelial cells tube formation in vitro. Accordingly, various angiogenic inhibitors are available for commercial acquisition.
Thermo Fisher Scientific offers interferon-β recombinant rat protein and interferon-γ recombinant mouse protein, but also interleukin-4 recombinant human protein. The latter has demonstrated angiostatic effects, and it can mediate inhibition of angiogenesis by associating with altered proangiogenic and antiangiogenic cytokines.
Immunosuppressant drug cyclosporin A and anti-fungal drug itraconazole synergistically inhibit endothelial cell tube formation and sprout formation. Under normoxic conditions, chemical compound pyrrolidine dithiocarbamate (PDTC) can inhibit the development of capillary like tubes.
Moreover, an overexpression of certain gain-of-function mutant forkhead box (FoxO) proteins (such as FoxO1 of FoxO3a) can also significantly inhibit tube formation in vitro. Therefore further experiments in this field are needed in order to gain more insight into the processes and factors guiding endothelial tube formation.