Validation of collagenous protein synthesis as an index for angiogenesis with the use of morphological methods

A method providing a biochemical index for the evaluation of promoters or inhibitors of angiogenesis in the chick chorioallantoic membrane (CAM) is here described and validated. This method is based on the determination of collagenous protein synthesis which takes place during new vessel formation. Validation was done by comparing collagenous protein synthesis to morphological methods of determining vascular density either by counting the number of vessels intersecting three concentric rings or by computer-assisted image analysis. Five compounds which promote or inhibit angiogenesis in the CAM were used for this purpose. The protein kinase C activator 4-beta-phorbol 12-myristate 13-acetate and alpha-thrombin increased collagenous protein synthesis and these results correlated with those obtained by using the two morphological methods mentioned above. Similarly, the inhibitors of angiogenesis, Ro318220, tricyclodecan-9-yl xanthate (D609), and 8,9-dihydroxy-7-methyl-benzo[b]quinolizinium bromide (GPA1734), reduced collagenous protein synthesis and vascular density (determined by image analysis or by counting the number of vessels intersecting three concentric rings) to a comparable degree. These results indicate that collagenous protein synthesis can be used as a reliable, reproducible, and unbiased index of angiogenesis in the chick chorioallantoic membrane.