The Rat Aorta Model of Angiogenesis: Methodology and Applications

Abstract

Angiogenesis can be studied in vitroby culturing rings of rat aorta in collagen gels under serum-free conditions. Ring-shaped expiants are obtained by cross sectioning the rat aorta under a dissecting microscope. The aortic rings are washed with serum-free MCDB 131 growth medium and embedded in collagen gels prepared in agarose culture wells. After gelation has occurred, each collagen gel is freed of the surrounding agarose support and transferred to a plastic culture well where it floats in 0.5 ml of serum-free medium. The cultures are kept in a humidified CO₂ incubator at 35.5°C. Aortic rings grown under these conditions generate branching microvessels in the absence of serum or exogenous growth factors. The angiogenic response can be modulated by adding soluble angiogenic agonists or antagonists to the culture medium. The solid phase in which the aortic rings are embedded can be modified by incorporating additional extracellular matrix molecules to the collagen solution before gelation. The neovessels arise primarily from the luminal cut edges of the expiants and are composed of endothelial cells surrounded by pericytes. The cultures contain fibroblasts which are the first cells to migrate out of the aortic wall. The angiogenic response starts at day 3–4 and ends at day 9–10. After it has formed, the vascular outgrowth undergoes a process of regression and remodeling with retraction of the small endothelial branches into the main stem of the microvessels. During this phase, pericytes increase in number by migrating and proliferating at the abluminal surface of the endothelium. Angiogenesis can be quantitated by direct visual counts of microvessels and by computer-assisted image analysis. The rat aorta model can be used to evaluate various aspects of the angiogenic process including autocrine/paracrine regulation of angiogenesis by cells of the vessel wall, modulation of angiogenesis by the extracellular matrix, stimulation of microvessel formation by angiogenic factors, and inhibition of angiogenesis.