An ex vivo angiogenesis assay utilizing commercial porcine carotidartery: Modification of the rat aortic ring assay

Abstract

The study of angiogenesis as a therapeutic target requires a reliable, physiologically relevant, and technically straightforward assay. An ex vivo assay bridges the gap between cell-based assays, which may not realistically represent the complex process of vessel sprouting, and in vivo assays, which are time consuming and expensive. Porcine carotid arteries provide an ideal tissue source for angiogenesis inhibitor screens due to their availability, physiological relevance and large size. 1.5 mm² fragments of porcine carotid arteries were incubated in 48-well culture plates and sandwiched between two 100 μl layers of Matrigel. Sprouting was observed from the explants and quantitated, using a digital imaging system, after two weeks of incubation. Histological analysis using Factor VIII-related antigen (von Willebrand Factor) as an endothelial cell-specific marker identified these sprouts, which were consistent with endothelial cell morphology, supporting the system as a model of angiogenesis. Accordingly, the angiogenesis inhibitors suramin, 2-methoxyestradiol, and the matrix metalloprotease inhibitor Batimastat were shown to completely inhibit sprouting at 50, 0.5, and 5.0 μg/ml, respectively and to have ED₅₀ values of 23, 0.15, and 0.14 μg/ml. This assay shows good reproducibility and eliminates animal to animal variation. The system should prove adaptable to other forms of angiogenic stimulation, ultimately making a variety of assays for angiogenesis available to laboratories of limited resources.