A novel assay of angiogenesis in the quail chorioallantoic membrane: stimulation by bFGF and inhibition by angiostatin according to fractal dimension and grid intersection.

In a novel assay of angiogenesis in the quail chorioallantoic membrane (CAM), we measured vascular pattern and angiogenic rate after homogeneous exposure of the entire vascular tree to recognized modulators of vessel growth. In comparison to phosphate-buffered saline (PBS)-treated controls, the vascular stimulator, basic fibroblast growth factor (bFGF or FGF-2), increased the rate of angiogenesis by a maximum of 72%, whereas a recently discovered angiogenic inhibitor, angiostatin, decreased the rate of vascular growth by a maximum of 68%. The perturbants were applied in PBS to the CAM of 7-day-old embryos (E7) cultured in petri dishes, and the embryos were cultured further until fixation at E8 or E9. For morphometry of the quasi-two-dimensional CAM vasculature, digital images of arterial endpoints from the middle region of the CAM were acquired in grayscale at a magnification of 10x, binarized to black/white, and skeletonized. The pattern of vessel branching was assessed by measurement of the fractal dimension (Df), and vessel density (rhov), with the method of grid intersection. Correlations between these two statistical techniques were linear (r2 ranged from 0.967 to 0.985). For skeletonized images at E9, Df and rhov of bFGF-treated samples were 1.55 +/- 0.01 and 782 +/- 26/cm2, respectively (relative to 1.49 +/- 0.02 and 583 +/- 60/cm2 for controls), and of angiostatin-treated samples, 1.43 +/- 0.02 and 424 +/- 74/cm2 (relative to 1.50 +/- 0.02 and 616 +/- 59/cm2 for controls). To establish normalization values for rates of angiogenesis, we analyzed untreated CAMs of E6 to E12. From E7 to E10 in skeletonized images, Df increased linearly from 1.37 +/- 0.01 to 1.54 +/- 0.01 and rhov from 311 +/- 67 to 746 +/- 124/cm2 (in both cases, r2 = 1.000). Thus, the rates of normal angiogenic growth as measured by Df and rhov were 0.06/day and 138/cm2-day, respectively. From E10 to E12, Df and rhov declined slightly. Differences between the vasculature of untreated and PBS-treated CAMs were statistically insignificant. In conclusion, vascular branching pattern and density in the quail CAM were stimulated by bFGF and inhibited by angiostatin. We quantified these changes with statistical significance by Df and rhov, which are expressed relative to the rates of normal developmental angiogenesis measured for the two parameters in untreated quail embryos. Copyright 1998 Academic Press.