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 homogen eous exposure of the entire vascular tree to recognized modulators of vessel growth. In comparison to phosphate-buffered saline (PBS)-treate d controls, the vascular stimulator, basic fibroblast growth factor (b FGF or FGF-2), increased the rate of angiogenesis by a maximum of 72%, whereas a recently discovered angiogenic inhibitor, angiostatin, decr eased 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 C AM were acquired in grayscale at a magnification of 10x, binarized to black/white, and skeletonized. The pattern of vessel branching was ass essed by measurement of the fractal dimension (Df), and vessel density (rho(v)), with the method of grid intersection. Correlations between these two statistical techniques were linear (r(2) ranged from 0.967 t o 0.985). For skeletonized images at E9, D-f and rho(v) of bFGF-treate d samples were 1.55 +/- 0.01 and 782 +/- 26/cm(2), respectively (relat ive to 1.49 +/- 0.02 and 583 +/- 60/cm(2) for controls), and of angios tatin-treated samples, 1.43 +/- 0.02 and 424 +/- 74/cm(2) (relative to 1.50 +/- 0.02 and 616 +/- 59/cm(2) for controls). To establish normal ization values for rates of angiogenesis, we analyzed untreated CAMs o f E6 to E12. From E7 to E10 in skeletonized images, Df increased linea rly from 1.37 +/- 0.01 to 1.54 +/- 0.01 and rho(v) from 311 +/- 67 to 746 +/- 124/cm(2) (in both cases, r(2) = 1.000). Thus, the rates of no rmal angiogenic growth as measured by D-f and rho(v) were 0.06/day and 138/cm(2)-day, respectively. From E10 to E12, D-f and rho(v) declined slightly. Differences between the vasculature of untreated and PBS-tr eated CAMs were statistically insignificant. In conclusion, vascular b ranching pattern and density in the quail CAM were stimulated by bFGF and inhibited by angiostatin. We quantified these changes with statist ical significance by D-f and rho(v), which are expressed relative to t he rates of normal developmental angiogenesis measured for the two par ameters in untreated quail embryos. (C) 1998 Academic Press.