Very low concentrations of rat plasma and rat serum stimulate angiogenesis in the rat aortic ring assay

Angiogenesis can be achieved in serum-free culture using the rat aortic ring assay developed by Nicosia et al.1 This is a reproducible and sensitive method of quantifying stimulatory or inhibitory effects of various substances on angiogenesis by measuring the amount of microvessel formation. Animal serum is known to contain various factors that can maintain the growth of endotheîial cells in culture. For this purpose, 10% fetal bovine serum is usually used. We studied the effect of low concentrations of rat plasma and rat serum on angiogenesis using the rat aortic ring assay as previously described. For quantification of the microvessels, digital pictures of the aortic rings were taken at day 0 and every 3 days thereafter until day 12. The images were downloaded into a computer and convened into gray scale. NIH Image software was used for analysis. Images from day 0 were subtracted from the following days. Integrated density of the subtracted images (sum of the gray values of all the microvessel pixels), which is a measure of the total surface area of the microvessels, was calculated and compared. Varying concentrations of rat plasma or rat serum ranging from 0.25% to 3% were added to MCDB 131 growth medium. The aortic rings embedded in fibrin gel were incubated with the media which were changed every 3 days. Both rat plasma and rat serum stimulated angiogenesis at all concentrations tested in a dose dependent manner. At 0.5% and 3% concentrations, rat plasma increased microvessel growth by about 125% and 250% respectively, while rat serum produced microvessel growth increments of about 500% and 2350% respectively. At plasma or serum concentrations above 1%, fibroblast proliferation is usually more intense and thus may significantly contribute to the image integrated density. This angiogenic effect of rat plasma and serum was not affected by heating up to 100°C for 10 min or overnight dialysis against 0.15 M NaCl using a regenerated cellulose membrane (Spectra/For 4) which has a molecular weight cut off of 12-14 kDa. The active fraction in the plasma is co-eluted with albumin after gel chromatography (Sepharose CL 4B). In conclusion, we have shown that very low concentrations of rat plasma and rat serum stimulates angiogenesis in the rat aortic ring assay. This potent angiogenic effect present in both plasma and serum may be distinct and probably cannot be accounted for by the properties of known growth factors. 1. Nicosia et al. Lab. Invest., 63: 115-122, 1990.