Quantitative Analysis of Angiogenesis Inhibition by Cleaved High Molecular Weight Kininogen Using VESGEN.

Angiogenesis requires specific molecular regulation of complex vascular branching morphology. The cleaved form of high molecular weight kininogen (HKa) has been shown to cause apoptosis of proliferating endothelial cells, and inhibit angiogenesis in vivo. However, critical morphological effects of HKa on the complex branching morphology of angiogenic vascular trees have not been defined. We used the quail chorioallantoic membrane (CAM) angiogenesis assay, in which angiogenesis agonists or antagonists cause uniform perturbation of angiogenesis throughout the entire vascular tree, to assess the effects of HKa on branching morphology. HKa (1.56 to 50 micrograms) was applied uniformly to the CAM, and its effect on angiogenesis determined after 24 hours of incubation. VESGEN vascular analysis software, currently under development at NASA Glenn Research Center, was used to quantify major vessel parameters in low-magnification images of vascular trees automatically extracted by VESGEN. Results derived from qualitative observation and initial quantification of two experiments demonstrates that HKa significantly inhibits vascular growth. Vessel length density (Lv) and branch point density (Br) in CAM specimens treated with 25 micrograms of HKa decreased by 23% and 31% respectively (24 ± 1 cm/cm² and 441 ± 43 /cm², compared to 31 ± 0 cm/cm² and 677 ± 51 /cm² in vehicle-treated controls; mean +/− S.E. for n = 3). The fractal dimension (Df) of skeletonized vascular images is a highly sensitive indicator of vessel density that ranges from 1.35 for strong angiogenesis inhibition, to 1.40 and 1.48 for controls and strong angiogenesis stimulation, respectively. D_f decreased to 1.35 ± 0.01 in the HKa-treated specimens, relative to 1.39 ± 0.00 in controls. However, thickening of large vessels in response to HKa resulted in a virtually equivalent vessel area density (Av) of 0.167 ± 0.012 cm²/cm², relative to 0.174 ± 0.015 cm²/cm² in controls. Further studies of the site-specific effects of HKa on branching morphology within the vascular tree are in progress. These results demonstrate that the antiangiogenic effects of HKa result in a unique antiangiogenic ’fingerprint’ vascular morphology in comparison to other angiogenesis inhibitors that include transforming growth factor-β1 and angiostatin.