Angiogenesis represents the formation of new blood vessels (either via sprouting or branching) from existing ones. This process is involved in tumor growth and metastasis, thus its inhibition is often a critical point of intervention in the treatment of malignancies.
Endothelial cells need to undertake four crucial steps in order to form new blood vessels. Those are degradation of adjacent matrix, migration toward the angiogenic stimuli, proliferation to create additional cells for the new blood vessels, as well as reorganization to form 3D vessel structures.
The development of angiogenesis assays was a pivotal step in the discovery of proangiogenic and antiangiogenic molecules. Among them, in vitro assays have been quantitative and expeditious as initial approximations that warrant further confirmation in vivo.
Characteristics of in vitro assays
A majority of endothelial cell assays utilize human umbilical vein endothelial cells or bovine aortic endothelial cells – not because these types of cells are good representatives of vascular conditions in vivo, but primarily because they are relatively easy to harvest from large blood vessels.
It must be emphasized that there are differences among endothelial cells from large and small blood vessels, as well as from different organs. Furthermore, endothelial cells that are used in laboratory conditions are basically always in a proliferative state rather than the normal quiescent state of the established vasculature.
Another issue is the environmental conditions in which endothelial cells are cultured. In the living organism, endothelial cells are exposed to a plethora of hemodynamic forces that activate multiple signaling pathways. On the other hand, endothelial cells in the laboratory are usually cultured in room air, which is hyperoxic compared with the in vivo oxygen tensions (especially in the microcirculation).
Furthermore, in vivo interaction between endothelial cells and other cells types (such as fibroblasts, pericytes and macrophages) is cumbersome to translate and simulate in vitro. Therefore in vitro angiogenesis assays should be viewed as a starting point rather than an endpoint for discovery.
Types of in vitro assays
There are a myriad of well-established in vitro methods for studying cell proliferation. The most reliable among them calculates proliferation by direct measurement of DNA synthesis, and thymidine incorporation assay is often cited as a reference model.
Thymidine Incorporation Assay BrdU offered by a company Thermo Fisher Scientific includes a nucleoside analog that is identified during active DNA synthesis by employing immunohistochemistry. This firm also developed Click-iT EdU Microplate Assay that utilizes nucleoside analog EdU (5-ethynyl-2'-deoxyuridine) and a detection method that does not necessitate DNA denaturation.
In some types of angiogenesis, endothelial cells degrade the basement membrane and migrate along chemical gradients previously established by proangiogenic growth factors. The transwell migration assay (a modification of the classical Boyden chamber assay) is frequently utilized to assess endothelial cell migration, which is one of the main in vitro assays in the repertoire of Thermo Fisher Scientific.
24- or 96-multiwell BD FluoroBlok insert (with 3 μm pore size) coated with human fibronectin was developed by a company BD Biosciences, and also serves as a cell migration assay. Other variants of migration assays include under-agarose assay, Teflon fence assay, wound healing assay and phagokinetic track assay.
One of the highly specific tests for angiogenesis is assessing the propensity of endothelial cells to form 3D structures (also known as tube formation). At the moment, the most popular tube formation assays involve plating human umbilical vein endothelial cells or bovine aortic endothelial cells with Matrigel.
The aortic ring angiogenesis assay is widely used in angiogenesis research due to its reliability and reproducibility. Other in vitro angiogenesis assays that cover all angiogenic functions (i.e. proliferation, migration, tube formation) include mouse metatarsal assay, embryoid assay and others.