Angiogenesis assays

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ANGIOGENIC ASSAYS In the effort to understand the underlying mechanisms of angiogenesis and in the search for new therapeutic modalities, in vitro and in vivo angiogenic assays represent important tools. In the following we will review some of the most commonly used in vitro methods and at the same time invite other researchers in the fields to come up with their experience and suggestions.

1.Cell proliferation Proliferation assays are based on direct cell counting, thymidine incorporation or staining techniques for cell proliferation or death and takes advantage of the proliferation of endothelial cells after stimulation with cytokines. The source of endothelial cells may influence the outcome of tests validating angiogenic activity, as endothelial cells originating from large vessels differ from endothelial cells from microvasculature and endothelial cells from different organs may have distinct properties. Another aspect to consider is that endothelial cells used in laboratory assays are in a proliferative phase, compared to endothelial cells in a mature resting vessel in a steady state with low levels of cell proliferation and migration.

2 Endothelial cell migration Migration assays are based on the ability of endothelial cells to migrate in response to chemotactic stimuli. Most assays use different variants of the Boyden chamber, where 2 compartments are separated by a filter membrane with a pore size allowing endothelial cells to pass by active migration. When endothelial cells are added to the top well, chemotactic solutions are placed in the lower well and cells that have migrated toward the chemotactic stimulus are counted after a period of incubation. The migrated cells on the other side of the membrane can be counted manually.

3. Cell matrix interactions The most commonly used assay to study cell-matrix interactions, is cell adhesion assay. Adhesion of ECs to extracellular matrix proteins such as collagen IV, laminin, vitronectin, and fibronectin is mediated by various surface receptors belonging to several families, such as integrins and cell surface proteoglycans. Interactions between matrix proteins and endothelial cells play important roles in cell survival, proliferation and migration.

4. Tube formation Another approach is to study the formation of capillary-like tubes when endothelial cells are cultured on Matrigel, a mixture of extracellular and basement membrane proteins, particularly laminin, prepared from Engelbreth-Holm-Swarm (EHS) mouse tumors. Tube formation during angiogenesis involves complex cell-cell and cell-matrix interactions. This method is fast, but it is not clear if the “tubules” really possess a lumen. It is reported that cells of non-endothelial origin, such as fibroblasts can form tubule-like strands on Matrigel. Results from this assay should therefore be interpreted with great caution.