The blood vessel system is essential for supplying cells with nutrients and oxygen, in addition to the removal of waste products. All cells in an organ are located in close proximity to this supportive system. To influence vessel growth and ensure their own sustenance, tumour cells release growth factors such as vascular endothelial growth factor, leading to tumour-directed vessel development. This process is called pathological angiogenesis, a development of new vessels from pre-existing vessels. Different angiostatic drugs can be applied to disrupt vessel growth and therefore limit tumour nutrition. Different in vitro and in vivo assays have been developed to investigate the effect of angiostatic drugs. A commonly used and well-known assay is the Regorafenib Matrigel assay, which is rather poorly characterized. Capillary-like structures with lumen have been described using this assay, although there is significant debate as to whether these structures actually contain patent lumina or not. Furthermore, non-endothelial cells such as fibroblasts and other cell types also form tubules on Matrigel. For this reason, the results need to be interpreted with caution and more than one assay should be taken into consideration. A further assay used to evaluate the efficacy of angiostatic drugs is the endothelial cell co-culture assay. This assay is based on a supportive mural cell layer, on which endothelial cells have the ability to form capillary-like structures after 7–14 days. Although this assay takes longer than the Matrigel assay, it provides a more physiological environment, with tubules growing on the supportive mural cell layer matrix. For the endothelial cell co-culture assay, a variety of different cell types have been employed as a supportive cell layer, including pulmonary artery smooth muscle cells, primary human mammary fibroblasts and human dermal fibroblasts. However, these sources of human tissue-derived cell are limited, and more accessible human or animal tissue-derived cell sources would be an advantage for endothelial cell co-culture assays. In the present approach, HUASMCs and ovine carotid-artery derived cells were investigated as accessible, supportive cell layers for endothelial cell co-culture assays. We evaluated the influence of cell numbers within the supportive cell layer, in addition to that of pro-angiogenic factors and anti-angiogenic factors, on vessel development. The presence of VEGF receptor-2 on the employed HUVEC cell lines as the tubule-forming units was also evaluated to determine any correlation with the amount of capillary-like structures formed in vitro. Blood vessels function to supply all cells of an organism with oxygen and nutrition. A tumour located in close proximity to this system and not exceeding a certain size is sufficiently supplied by diffusion. A larger tumour needs to recruit additional blood vessels by angiogenesis. The tumour itself can initiate these processes by releasing growth factors such as VEGF, and by consequently changing the balance between pro- and antiangiogenic molecules. This results in pathological angiogenesis in the direction of the tumour.